Anti-npr1 antibodies and uses thereof

ABSTRACT

The present invention provides monoclonal antibodies that bind to the natriuretic peptide receptor 1 (NPR1) protein, and methods of use thereof. In various embodiments of the invention, the antibodies are fully human antibodies that bind to NPR1. In some embodiments, the antibodies of the invention are useful for activating NPR1 activity, thus providing a means of treating or preventing a disease, disorder or condition associated with NPR1 in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 62/749,557, filed on Oct. 23, 2018, and U.S.provisional application No. 62/755,720, filed on Nov. 5, 2018, thedisclosures of which are herein incorporated by reference in itsentirety.

SEQUENCE STATEMENT

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 16, 2019, isnamed SequenceList_10471 US01.TXT and is 99.3 kilobytes in size.

FIELD OF THE INVENTION

The present invention is related to antibodies and antigen-bindingfragments of antibodies that specifically bind to natriuretic peptidereceptor 1 (NPR1), and therapeutic and diagnostic methods of using thoseantibodies.

BACKGROUND OF THE INVENTION

Natriuretic peptide receptor 1 (NPR1; also known as NPR-A) belongs tothe cell-surface family of the guanylyl cyclase receptors, enzymes thatcatalyze the conversion of GTP into cyclic GMP. NPR1 is highly expressedin kidney, lungs, adrenal, vasculature, brain, liver, endothelial andadipose tissues and at lower levels in the heart. It is activated bybinding to atrial natriuretic peptide (ANP) or brain natriuretic peptide(BNP). NPR1 activation and signaling stimulate many physiologicresponses involving many tissues. The ANP-NPR1 system has been wellstudied for its role in vasorelaxation, natriuresis, diuresis,endothelial permeability and in non-cardiovascular functions likelipolysis and immune cell functions (Potter 2011, Pharmacol. Ther. 130:71-82). Activation of NPR1 leads to natriuresis (excretion of salt bykidneys) and lowers blood pressure.

Monoclonal antibodies to NPR1 were first described by Kitano et al in1995 in Immunol. Lett. 47: 215-22. Activating or agonist anti-NPR1antibodies are disclosed in, for example, US Patent/Publication Nos.9090695, and 20160168251, and in WO2010065293.

Fully human antibodies that specifically bind to NPR1 protein with highaffinity and activate it could be important in the prevention andtreatment of, e.g., hypertension, obesity and heart failure.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies and antigen-binding fragmentsthereof that specifically bind the natriuretic peptide receptor 1 (NPR1)protein. In certain embodiments, the anti-NPR1 antibodies are fullyhuman antibodies that bind to NPR1 with high affinity and activate NPR1or stabilize the activated conformation. The antibodies of the presentinvention are useful, inter alia, for activating or increasing theactivity of NPR1 protein. In certain embodiments, the antibodies areuseful in preventing, treating or ameliorating at least one symptom orindication of a NPR1-associated disease or disorder in a subject. Incertain embodiments, the antibodies may be administered prophylacticallyor therapeutically to a subject having or at risk of having aNPR1-associated disease or disorder. In specific embodiments, theantibodies are used to decrease systemic blood pressure in a subjectsuffering from high blood pressure. Such antibodies can be used astherapy for a disorder such as heart failure when administered to asubject in need thereof.

In certain embodiments, the antibodies bind to NPR1 in the presence orabsence of atrial natriuretic peptide (ANP) or brain natriuretic peptide(BNP), i.e., the antibodies are “peptide-independent binders.” Suchantibodies are advantageous as they can be used to bind and activateNPR1 irrespective of differing concentrations of endogenous ligand. Suchantibodies when administered to a patient in need thereof can beadvantageously used to avoid patient-to-patient variability (withrespect to ligand concentration) in treatment. Further, the antibodiesdisclosed herein bind to NPR1 with high affinity and have improvedpharmacokinetic properties (as compared to standard-of-care drugs). Theantibodies showed a t ½ of up to 11 days in mice at a dose of 25 mg/kg.The antibodies are efficacious in lowering the blood pressure andmaintaining the lowered pressure for as long as 28 days whenadministered to a subject in need thereof. A single dose of an antibodyof the present invention led to sustained reduction in blood pressure.Such antibodies can be used to provide superior efficacy, along withless frequent dosing, in a subject with a NPR1-associated disease ordisorder (e.g., hypertension).

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to increase persistence in the host or to eliminateresidual effector functions (Reddy et al., 2000, J. Immunol.164:1925-1933). In certain embodiments, the antibodies may bebispecific.

In a first aspect, the present invention provides isolated recombinantmonoclonal antibodies or antigen-binding fragments thereof that bindspecifically to NPR1.

In some embodiments, the present invention provides an isolated antibodyor antigen-binding fragment thereof that binds specifically tonatriuretic peptide receptor 1 (NPR1) protein, wherein the antibody orantigen-binding fragment thereof interacts with one or more amino acidscontained within the extracellular domain of NPR1 (amino acids 29-347 ofSEQ ID NO: 194), as determined by hydrogen/deuterium exchange, andwherein the antibody or antigen-binding fragment thereof: (i) binds tocells expressing human NPR1 in the presence or absence of atrialnatriuretic peptide (ANP); and/or (ii) binds to NPR1 and activates NPR1.

In some embodiments, the antibodies are fully human monoclonalantibodies.

Exemplary anti-NPR1 antibodies of the present invention are listed inTables 1 and 2 herein. Table 1 sets forth the amino acid sequenceidentifiers of the heavy chain variable regions (HCVRs), light chainvariable regions (LCVRs), heavy chain complementarity determiningregions (HCDRs) (HCDR1, HCDR2 and HCDR3), and light chaincomplementarity determining regions (LCDRs) (LCDR1, LCDR2 and LCDR3) ofexemplary antibodies. Table 2 sets forth the nucleic acid sequenceidentifiers of the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 andLCDR3 of the exemplary antibodies.

The present invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HCVR comprising an amino acid sequence selectedfrom any of the HCVR amino acid sequences listed in Table 1, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an LCVR comprising an amino acid sequenceselected from any of the LCVR amino acid sequences listed in Table 1, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCVR and an LCVR amino acid sequencepair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listedin Table 1 paired with any of the LCVR amino acid sequences listed inTable 1. According to certain embodiments, the present inventionprovides antibodies, or antigen-binding fragments thereof, comprising anHCVR/LCVR amino acid sequence pair contained within any of the exemplaryanti-NPR1 antibodies listed in Table 1. In certain embodiments, theHCVR/LCVR amino acid sequence pair is selected from one of SEQ ID NOs:2/10 (e.g., mAb22033), and 66/74 (e.g., mAb22810).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a HCVR and a LCVR, said HCVR comprising anamino acid sequence listed in Table 1 having no more than twelve aminoacid substitutions, and/or said LCVR comprising an amino acid sequencelisted in Table 1 having no more than ten amino acid substitutions. Forexample, the present invention provides antibodies or antigen-bindingfragments thereof comprising a HCVR and a LCVR, said HCVR comprising anamino acid sequence listed in Table 1, said amino acid sequence havingone, two, three, four, five, six, seven, eight, nine, ten, eleven ortwelve amino acid substitutions. In another example, the presentinvention provides antibodies or antigen-binding fragments thereofcomprising a HCVR and a LCVR, said LCVR comprising an amino acidsequence listed in Table 1, said amino acid sequence having one, two,three, four, five, six, seven, eight, nine or ten amino acidsubstitutions. In one embodiment, the present invention providesanti-NPR1 antibodies or antigen-binding fragments thereof comprising aHCVR and a LCVR, said HCVR comprising an amino acid sequence listed inTable 1, said amino acid sequence having at least one amino acidsubstitution, and/or said LCVR comprising an amino acid sequence listedin Table 1, said amino acid sequence having at least one amino acidsubstitution.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising anamino acid sequence selected from any of the HCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising anamino acid sequence selected from any of the HCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising anamino acid sequence selected from any of the HCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR1 (LCDR1) comprising anamino acid sequence selected from any of the LCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR2 (LCDR2) comprising anamino acid sequence selected from any of the LCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR3 (LCDR3) comprising anamino acid sequence selected from any of the LCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequencepair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequenceslisted in Table 1 paired with any of the LCDR3 amino acid sequenceslisted in Table 1. According to certain embodiments, the presentinvention provides antibodies, or antigen-binding fragments thereof,comprising an HCDR3/LCDR3 amino acid sequence pair contained within anyof the exemplary anti-NPR1 antibodies listed in Table 1. In certainembodiments, the HCDR3/LCDR3 amino acid sequence pair is selected fromthe group consisting of SEQ ID NOs: 8/16 (e.g., mAb22033), and 72/80(e.g., mAb22810).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a HCVR and a LCVR, said HCVR comprisingHCDR1 comprising an amino acid sequence differing from an amino acidsequence listed in Table 1 by 1 amino acid, HCDR2 comprising an aminoacid sequence differing from an amino acid sequence listed in Table 1 by1 amino acid, and HCDR3 comprising an amino acid sequence differing froman amino acid sequence listed in Table 1 by 1 amino acid. In certainembodiments, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a HCVR and a LCVR, saidLCVR comprising LCDR1 comprising an amino acid sequence differing froman amino acid sequence listed in Table 1 by 1 amino acid, LCDR2comprising an amino acid sequence differing from an amino acid sequencelisted in Table 1 by 1 amino acid, and LCDR3 comprising an amino acidsequence differing from an amino acid sequence listed in Table 1 by 1amino acid. For example, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a HCVR and a LCVR, saidHCVR comprising HCDR1 comprising an amino acid sequence of SEQ ID NO: 4or an amino acid sequence differing from SEQ ID NO: 4 by 1 amino acid,HCDR2 comprising an amino acid sequence of SEQ ID NO: 6 or an amino acidsequence differing from SEQ ID NO: 6 by 1 amino acid, and HCDR3comprising an amino acid sequence of SEQ ID NO: 8 or an amino acidsequence differing from SEQ ID NO: 8 by 1 amino acid. In anotherexemplary embodiment, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a HCVR and a LCVR, saidLCVR comprising LCDR1 comprising an amino acid sequence of SEQ ID NO: 12or an amino acid sequence differing from SEQ ID NO: 12 by 1 amino acid,LCDR2 comprising an amino acid sequence of SEQ ID NO: 14 or an aminoacid sequence differing from SEQ ID NO: 14 by 1 amino acid, and LCDR3comprising an amino acid sequence of SEQ ID NO: 16 or an amino acidsequence differing from SEQ ID NO: 16 by 1 amino acid.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a set of six CDRsHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of theexemplary antibodies listed in Table 1. In certain embodiments, theHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selectedfrom the group consisting of SEQ ID NOs: 4-6-8-12-14-16 (e.g.,mAb22033), and 68-70-72-76-78-80 (e.g., mAb22810).

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a set of six CDRsHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR aminoacid sequence pair as defined by any of the exemplary antibodies listedin Table 1. For example, the present invention includes antibodies, orantigen-binding fragments thereof, comprising theHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set containedwithin an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 2/10 (e.g., mAb22033), and 66/74 (e.g.,mAb22810). Methods and techniques for identifying CDRs within HCVR andLCVR amino acid sequences are well known in the art and can be used toidentify CDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

In certain embodiments, the present invention includes an antibody orantigen-binding fragment thereof that binds specifically to NPR1,wherein the antibody or antigen-binding fragment thereof comprises threeheavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 andHCDR3) contained within a heavy chain variable region (HCVR) and threelight chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chainvariable region (LCVR), wherein the HCVR comprises: (i) an amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 18, 34,50, 66, 82, 98, 114, 130, 146, 162, and 178; (ii) an amino acid sequencehaving at least 90% identity to the amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130,146, 162, and 178; (iii) an amino acid sequence having at least 95%identity to the amino acid sequence selected from the group consistingof SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178;or (iv) an amino acid sequence selected from the group consisting of SEQID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178, saidamino acid sequence having no more than 12 amino acid substitutions; andthe LCVR comprises: (a) an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154,170, and 186; (b) an amino acid sequence having at least 90% identity tothe amino acid sequence selected from the group consisting of SEQ IDNOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and 186; (c) anamino acid sequence having at least 95% identity to the amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 74, 90, 106, 122, 138, 154, 170, and 186; or (d) an amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 74, 90, 106, 122, 138, 154, 170, and 186, said amino acid sequencehaving no more than 10 amino acid substitutions.

In certain preferred embodiments, the present invention includesantibodies that bind specifically to NPR1 in an agonist manner, i.e.,potentiate or induce NPR1 binding and/or activity.

The present invention includes anti-NPR1 antibodies having a modifiedglycosylation pattern. In some embodiments, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In certain embodiments, the present invention provides antibodies andantigen-binding fragments thereof that exhibit pH-dependent binding toNPR1. For example, the present invention includes antibodies andantigen-binding fragment thereof that bind NPR1 with higher affinity atneutral pH than at acidic pH (i.e., reduced binding at acidic pH).

The present invention also provides for antibodies and antigen-bindingfragments thereof that compete for specific binding to NPR1 with anantibody or antigen-binding fragment thereof comprising the CDRs of aHCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an aminoacid sequence selected from the HCVR and LCVR sequences listed in Table1.

The present invention also provides antibodies and antigen-bindingfragments thereof that cross-compete for binding to NPR1 with areference antibody or antigen-binding fragment thereof comprising theCDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVR eachhas an amino acid sequence selected from the HCVR and LCVR sequenceslisted in Table 1.

The present invention also provides antibodies and antigen-bindingfragments thereof that bind to the same epitope as a reference antibodyor antigen-binding fragment thereof comprising three CDRs of a HCVR andthree CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acidsequence selected from the HCVR and LCVR sequences listed in Table 1.

The present invention also provides isolated antibodies andantigen-binding fragments thereof that increase or stabilize NPR1binding to its ligand (e.g., ANP or BNP). In some embodiments, theantibody or antigen-binding fragment thereof that activates NPR1 bindingto ANP may bind to the same epitope on NPR1 as ANP or may bind to adifferent epitope on NPR1 as ANP.

In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention are bispecific comprising a first bindingspecificity to a first epitope of NPR1 and a second binding specificityto a second epitope of NPR1 wherein the first and second epitopes aredistinct and non-overlapping.

In certain embodiments, the present invention provides an isolatedantibody or antigen-binding fragment thereof that has one or more of thefollowing characteristics: (a) is a fully human monoclonal antibody; (b)binds to monomeric human NPR1 in the absence of ANP and/or BNP at 25° C.and at 37° C. with a dissociation constant (K_(D)) of less than 690 nM,as measured in a surface plasmon resonance assay; (c) binds to dimerichuman NPR1 in the absence of ANP or BNP at 25° C. and at 37° C. with aK_(D) of less than 42 nM, as measured in a surface plasmon resonanceassay; (d) binds to human NPR1 complexed to ANP at 25° C. and 37° C.with a K_(D) of less than 80 nM, as measured in a surface plasmonresonance assay; (e) binds to human NPR1 complexed to BNP at 25° C. and37° C. with a K_(D) of less than 20 nM, as measured in a surface plasmonresonance assay; (f) binds to monomeric monkey NPR1 in the absence ofANP and/or BNP at 25° C. and 37° C. with a K_(D) of less than 365 nM, asmeasured in a surface plasmon resonance assay; (g) binds to dimericmonkey NPR1 in the absence of ANP or BNP at 25° C. and at 37° C. with aK_(D) of less than 30 nM, as measured in a surface plasmon resonanceassay; (h) binds to monkey NPR1 complexed to ANP at 25° C. and 37° C.with a K_(D) of less than 10 nM, as measured in a surface plasmonresonance assay; (i) binds to monkey NPR1 complexed to BNP at 25° C. and37° C. with a K_(D) of less than 10 nM, as measured in a surface plasmonresonance assay; (j) does not bind to mouse NPR1; (k) binds to cellsexpressing human NPR1 (without ANP) or NPR1-complexed to ANP with a EC₅₀less than 5 nM; (l) activates NPR1 with a EC₅₀ of less than 385 nM, asmeasured in a calcium flux cell-based bioassay; (m) reduces the systemicblood pressure when administered to normotensive and hypertensive mice,wherein the reduction in systemic and mean arterial blood pressureslasts for up to 28 days upon administration of a single dose; (n)improves glucose tolerance when administered to diet-induced obese mice;and (o) comprises a HCVR comprising an amino acid sequence selected fromthe group consisting of HCVR sequence listed in Table 1 and a LCVRcomprising an amino acid sequence selected from the group consisting ofLCVR sequences listed in Table 1.

In a second aspect, the present invention provides nucleic acidmolecules encoding anti-NPR1 antibodies or portions thereof. Forexample, the present invention provides nucleic acid molecules encodingany of the HCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs HCDR1-HCDR2-HCDR3),wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined byany of the exemplary antibodies listed in Table 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs LCDR1-LCDR2-LCDR3),wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined byany of the exemplary antibodies listed in Table 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 1,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-NPR1 antibody listed in Table1.

In a related aspect, the present invention provides recombinantexpression vectors capable of expressing a polypeptide comprising aheavy and/or light chain variable region of an antibody. For example,the present invention includes recombinant expression vectors comprisingany of the nucleic acid molecules mentioned above, i.e., nucleic acidmolecules encoding any of the HCVR, LCVR, and/or CDR sequences as setforth in Table 2. In certain embodiments, the present invention providesexpression vectors comprising: (a) a nucleic acid molecule comprising anucleic acid sequence encoding a HCVR of an antibody that binds NPR1,wherein the HCVR comprises an amino acid sequence selected from thegroup consisting of sequences listed in Table 1; and/or (b) a nucleicacid molecule comprising a nucleic acid sequence encoding a LCVR of anantibody that binds NPR1, wherein the LCVR comprises an amino acidsequence selected from the group consisting of sequences listed inTable 1. Also included within the scope of the present invention arehost cells into which such vectors have been introduced, as well asmethods of producing the antibodies or portions thereof by culturing thehost cells under conditions permitting production of the antibodies orantibody fragments, and recovering the antibodies and antibody fragmentsso produced. In certain embodiments, the host cells comprise a mammaliancell or a prokaryotic cell. In certain embodiments, the host cell is aChinese Hamster Ovary (CHO) cell or an Escherichia coli (E. coli) cell.In certain embodiments, the present invention provides methods ofproducing an antibody or antigen-binding fragment thereof of theinvention, the methods comprising introducing into a host cell anexpression vector comprising a nucleic acid sequence encoding a HCVRand/or LCVR of an antibody or antigen-binding fragment thereof of theinvention operably linked to a promoter; culturing the host cell underconditions favorable for expression of the nucleic acid sequence; andisolating the antibody or antigen-binding fragment thereof from theculture medium and/or host cell. The isolated antibody orantigen-binding fragment thereof may be purified using any of themethods known in prior art.

In a third aspect, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of at least onerecombinant monoclonal antibody or antigen-binding fragment thereof thatspecifically binds NPR1 and a pharmaceutically acceptable carrier. In arelated aspect, the invention features a composition that is acombination of an anti-NPR1 antibody and a second therapeutic agent. Inone embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-NPR1 antibody. Exemplary agentsthat may be advantageously combined with an anti-NPR1 antibody include,without limitation, other agents that bind and/or activate NPR1 activity(including other antibodies or antigen-binding fragments thereof, etc.)and/or agents which do not directly bind NPR1 but nonetheless treat orameliorate at least one symptom or indication of a NPR1-associateddisease or disorder (disclosed elsewhere herein). Additional combinationtherapies and co-formulations involving the anti-NPR1 antibodies of thepresent invention are disclosed elsewhere herein.

In a fourth aspect, the invention provides therapeutic methods fortreating a disease or disorder associated with NPR1 in a subject usingan anti-NPR1 antibody or antigen-binding portion of an antibody of theinvention, wherein the therapeutic methods comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising an antibody or antigen-binding fragment of an antibody of theinvention to the subject in need thereof. The disorder treated is anydisease or condition that is improved, ameliorated, inhibited orprevented by potentiation of NPR1 activity (e.g., hypertension). Incertain embodiments, the invention provides methods to prevent, or treata NPR1-associated disease or disorder comprising administering atherapeutically effective amount of an anti-NPR1 antibody orantigen-binding fragment thereof of the invention to a subject in needthereof. In some embodiments, the antibody or antigen-binding fragmentthereof may be administered prophylactically or therapeutically to asubject having or at risk of having a NPR1-associated disease ordisorder. In certain embodiments, the antibody or antigen-bindingfragment thereof the invention is administered in combination with asecond therapeutic agent to the subject in need thereof. The secondtherapeutic agent may be selected from the group consisting of analdosterone antagonist, an alpha-adrenergic blocker, an angiotensinconverting enzyme (ACE) inhibitor, an arteriolar vasodilator, anautonomic ganglionic vasodilator, a beta-adrenergic blocker, acatecholamine-depleting sympatholytic, a central alpha-2 adrenergicagonist, a calcium channel blocker, a diuretic, a renin inhibitor, ananti-coagulant, an anti-platelet agent, a cholesterol lowering agent, avasodilator, digitalis, surgery, an implantable device, anti-tumortherapy, insulin, a GLP1 agonist, metformin, dialysis, bone marrowstimulant, hemofiltration, a lifestyle modification, a dietarysupplement and any other drug or therapy known in the art. In certainembodiments, the second therapeutic agent may be an agent that helps tocounteract or reduce any possible side effect(s) associated with anantibody or antigen-binding fragment thereof of the invention, if suchside effect(s) should occur. The antibody or fragment thereof may beadministered subcutaneously, intravenously, intradermally,intraperitoneally, orally, or intramuscularly. The antibody or fragmentthereof may be administered at a dose of about 0.1 mg/kg of body weightto about 100 mg/kg of body weight of the subject. In certainembodiments, an antibody of the present invention may be administered atone or more doses comprising between 10 mg to 600 mg.

The present invention also includes use of an anti-NPR1 antibody orantigen-binding fragment thereof of the invention in the manufacture ofa medicament for the treatment of a disease or disorder that wouldbenefit from the activation of NPR1 binding and/or activity.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of selected anti-NPR1 agonist antibodies onsystolic blood pressure in normotensive NPR1^(hu/hu) mice. Telemeterednormotensive NPR1^(hu/hu) mice were randomized into groups based off ofbody weight. Animals were given a single 25 mg/kg subcutaneous injectionof an NPR1 agonist antibody or PBS control as described in Table 30. Allvalues are mean change from baseline for days 3-7±SEM, n=3-9 per group.Statistics—one-way ANOVA with Dunnett's; *p<0.05 vs. PBS control.

FIG. 2 shows the effect of anti-NPR1 antibody mAb22033 on systolic bloodpressure in normotensive NPR1^(hu/hu) mice. Telemetered normotensiveNPR1^(hu/hu) mice were randomized into five groups of equal body weightand given a single subcutaneous injection of mAb22033 at the doseslisted in Table 32. An IgG4 antibody was used as isotype control. Allvalues are mean±SEM, n=4-6 per group. Statistics—two-way ANOVA withDunnett's.

FIG. 3 shows the effect of anti-NPR1 antibody mAb22033 on diastolicblood pressure in normotensive NPR1^(hu/hu) mice. Telemeterednormotensive NPR1^(hu/hu) mice were randomized into five groups of equalbody weight and given a single subcutaneous injection of mAb22033 at thedoses listed in Table 32. An IgG4 antibody was used as isotype control.All values are mean±SEM, n=4-6 per group. Statistics—two-way ANOVA withDunnett's.

FIG. 4 shows the effect of anti-NPR1 antibody mAb22033 on heart rate innormotensive NPR1^(hu/hu) mice. Telemetered normotensive NPR1^(hu/hu)mice were randomized into five groups of equal body weight and given asingle subcutaneous injection of mAb22033 at the doses listed in Table32. An IgG4 antibody was used as isotype control. All values aremean±SEM, n=4-6 per group. Statistics—two-way ANOVA with Dunnett's.

FIG. 5 shows the effect of anti-NPR1 antibody mAb22033 on mean arterialblood pressure in normotensive NPR1^(hu/hu) mice. Telemeterednormotensive NPR1^(hu/hu) mice were randomized into five groups of equalbody weight and given a single subcutaneous injection of mAb22033 at thedoses listed in Table 32. An IgG4 antibody was used as isotype control.All values are mean±SEM, n=4-6 per group. Statistics—two-way ANOVA withDunnett's.

FIGS. 6A and 6B show the effect of anti-NPR1 antibody mAb22033 on leftventricular function in normotensive NPR1^(hu/hu) mice. (FIG. 6A) Endsystolic volume (%); and (FIG. 6B) End diastolic volume (%) intelemetered normotensive NPR1^(hu/hu) mice randomized into five groupsof equal body weight and given a single subcutaneous injection ofmAb22033 at the doses listed in Table 32. An IgG4 antibody was used asisotype control. Echocardiography was performed on anesthetized mice inthe short axis on day 28 post-dose using a high frequency ultrasoundsystem and probe. All values are mean±SEM, n=6-7 per group.Statistics—one-way ANOVA with Dunnett's; *p<0.05 vs. IgG4 isotypecontrol.

FIGS. 7A and 7B show the effect of anti-NPR1 antibody mAb22033 on leftventricular function in normotensive NPR1^(hu/hu) mice. (FIG. 7A)Fractional shortening (%); and (FIG. 7B) Ejection fraction (%) intelemetered normotensive NPR1^(hu/hu) mice randomized into five groupsof equal body weight and given a single subcutaneous injection ofmAb22033 at the doses listed in Table 32. An IgG4 antibody was used asisotype control. Echocardiography was performed on anesthetized mice inthe short axis on day 28 post-dose using a high frequency ultrasoundsystem and probe. All values are mean±SEM, n=6-7 per group.Statistics—one-way ANOVA with Dunnett's.

FIG. 8 shows the effect of a single dose of 2 anti-NPR1 antibodies onsystolic blood pressure in hypertensive NPR1^(hu/hu) mice. Telemeteredhypertensive NPR1^(hu/hu) mice were randomized into six groups of equalsystolic blood pressures and administered a single subcutaneous dose ofmAb22033 or mAb22810 at the doses listed in Table 36. An IgG4 antibodywas used as isotype control. All values are mean±SEM, n=4-6 per group.Statistics—two-way ANOVA with Dunnett's; *p<0.05 mAb22033 25 mg/kg vs.control; # p<0.05 mAb22033 5 mg/kg vs. control; !p<0.05 mAb22810 19mg/kg vs. control.

FIG. 9 shows the effect of a single dose of 2 anti-NPR1 antibodies ondiastolic blood pressure in hypertensive NPR1^(hu/hu) mice. Telemeteredhypertensive NPR1^(hu/hu) mice were randomized into six groups of equalsystolic blood pressures and administered a single subcutaneous dose ofmAb22033 or mAb22810 at the doses listed in Table 36. An IgG4 antibodywas used as isotype control. All values are mean±SEM, n=3-6 per group.Statistics—two-way ANOVA with Dunnett's;*p<0.05 mAb22033 25 mg/kg vs.control; # p<0.05 mAb22033 5 mg/kg vs. control; !p<0.05 mAb22810 19mg/kg vs. control; % p<0.05 mAb22810 5 mg/kg vs. control.

FIG. 10 shows the effect of a single dose of 2 anti-NPR1 antibodies onheart rate in hypertensive NPR1^(hu/hu) mice. Telemetered hypertensiveNPR1^(hu/hu) mice were randomized into six groups of equal systolicblood pressures and administered a single subcutaneous dose of mAb22033or mAb22810 at the doses listed in Table 36. An IgG4 antibody was usedas isotype control. All values are mean±SEM, n=3-6 per group.Statistics—two-way ANOVA with Dunnett's; *p<0.05 mAb22033 25 mg/kg vs.control; # p<0.05 mAb22033 5 mg/kg vs. control.

FIG. 11 shows the effect of a single dose of 2 anti-NPR1 antibodies onmean arterial blood pressure in hypertensive NPR1^(hu/hu) mice.Telemetered hypertensive NPR1^(hu/hu) mice were randomized into sixgroups of equal systolic blood pressures and administered a singlesubcutaneous dose of mAb22033 or mAb22810 at the doses listed in Table36. An IgG4 antibody was used as isotype control. All values aremean±SEM, n=3-6 per group. Statistics—two-way ANOVA with Dunnett's;*p<0.05 mAb22033 25 mg/kg vs. control; # p<0.05 mAb22033 5 mg/kg vs.control; !p<0.05 mAb22810 19 mg/kg vs. control.

FIG. 12 shows the effect of single and repeated doses of an anti-NPR1antibody on systolic blood pressure in hypertensive NPR1^(hu/hu) mice.Telemetered hypertensive NPR1^(hu/hu) mice were randomized into fivegroups of equal systolic blood pressures and administered either asingle subcutaneous dose or twice weekly for 3 weeks of mAb22033 at thedoses listed in Table 40. An IgG4 antibody was used as isotype control.All values are mean±SEM, n=3-6 per group. The arrows represent the dosesadministered to the mice. Statistics—two-way ANOVA with Dunnett's;*p<0.05 mAb22033 25 mg/kg vs. control; # p<0.05 mAb22033 5 mg/kg vs.control; !p<0.05 mAb22033 50 mg/kg vs. control.

FIG. 13 shows the effect of single and repeated doses of an anti-NPR1antibody on diastolic blood pressure in hypertensive NPR1^(hu/hu) mice.Telemetered hypertensive NPR1^(hu/hu) mice were randomized into fivegroups of equal systolic blood pressures and administered either asingle subcutaneous dose or twice weekly for 3 weeks of mAb22033 at thedoses listed in Table 40. An IgG4 antibody was used as isotype control.All values are mean±SEM, n=3-6 per group. The arrows represent the dosesadministered to the mice. Statistics—two-way ANOVA with Dunnett's.

FIG. 14 shows the effect of single and repeated doses of an anti-NPR1antibody on heart rate in hypertensive NPR1^(hu/hu) mice. Telemeteredhypertensive NPR1^(hu/hu) mice were randomized into five groups of equalsystolic blood pressures and administered either a single subcutaneousdose or twice weekly for 3 weeks of mAb22033 at the doses listed inTable 40. An IgG4 antibody was used as isotype control. All values aremean±SEM, n=3-6 per group. The arrows represent doses administered tothe mice. Statistics—two-way ANOVA with Dunnett's.

FIG. 15 shows the effect of single and repeated doses of an anti-NPR1antibody on mean arterial blood pressure in hypertensive NPR1^(hu/hu)mice. Telemetered hypertensive NPR1^(hu/hu) mice were randomized intofive groups of equal systolic blood pressures and administered either asingle subcutaneous dose or twice weekly for 3 weeks of mAb22033 at thedoses listed in Table 40. An IgG4 antibody was used as isotype control.All values are mean±SEM, n=3-6 per group. The arrows represent dosesadministered to the mice. Statistics—two-way ANOVA with Dunnett's;*p<0.05 mAb22033 25 mg/kg vs. control; # p<0.05 mAb22033 5 mg/kg vs.control; !p<0.05 mAb22033 50 mg/kg vs. control.

FIGS. 16A and 16B show the effects of single and repeated doses of ananti-NPR1 antibody on cardiac function in hypertensive NPR1^(hu/hu)mice. (FIG. 16A) end systolic volume %; and (FIG. 16B) end diastolicvolume % in telemetered hypertensive NPR1^(hu/hu) mice randomized intofive groups of equal systolic blood pressures and administered either asingle subcutaneous dose or twice weekly for 3 weeks of mAb22033 at thedoses listed in Table 40. An IgG4 antibody was used as isotype control.Echocardiography was performed on anesthetized mice in the short axis onday 21 post-dose using a high frequency ultrasound system and probe. Allvalues are mean±SEM, n=5-6 per group. Statistics—one-way ANOVA withDunnett's.

FIGS. 17A and 17B show the effects of single and repeated doses of ananti-NPR1 antibody on cardiac function in hypertensive NPR1^(hu/hu)mice. (FIG. 17A) Fractional shortening (%); and (FIG. 17B) Ejectionfraction (%) in telemetered hypertensive NPR1^(hu/hu) mice randomizedinto five groups of equal systolic blood pressures and administeredeither a single subcutaneous dose or twice weekly for 3 weeks ofmAb22033 at the doses listed in Table 40. An IgG4 antibody was used asisotype control. Echocardiography was performed on anesthetized mice inthe short axis on day 21 post-dose using a high frequency ultrasoundsystem and probe. All values are mean±SEM, n=5-6 per group.Statistics—one-way ANOVA with Dunnett's.

FIG. 18A shows changes in body weight following administration ofmAb22810 NPR1 agonist mAb, hFc.FGF21 or an isotype control mAb. FIG. 18Band FIG. 18C show total fat and total lean mass, respectively, after sixweeks of treatment as measured by EchoMRI. NPR1 humanized mice were madeobese by placing them on a 60% high-fat diet for 10 weeks. Followingthis period, mice were randomized into three groups of equal body weightand given a subcutaneous injection of treatments at the doses andfrequencies listed in Table 44. A human IgG4 antibody was used asisotype control. All values are mean±SEM, n=10 per group. *=P<0.05 vsisotype control; **=P<0.01 vs isotype control. Statistics by two-wayANOVA+Tukey for the body weights, one-way ANOVA+Bonferonni for the fatand lean mass.

FIGS. 19A, 19B, and 19C show changes in VO₂ (FIG. 19A), VCO₂ (FIG. 19B)or Energy Expenditure (FIG. 19C) broken down as the average of eachday/night cycle after one week of treatment with either mAb22810 NPR1agonist mAb, hFc.FGF21 or an isotype control mAb. After one week oftreatment, mice from each group were placed in a Columbia Instrumentsmetabolic cage system (CLAMS) for one week to record metabolicparameters. Mice were acclimated to the cages for one week prior tomeasurement to minimize stress. A human IgG4 antibody was used asisotype control. All values are mean±SEM, n=5-6 per group. **=P<0.01 vsisotype control, ***=P<0.001 vs isotype control. Statistics by one-wayANOVA+Bonferonni.

FIG. 20A shows changes in glucose tolerance as measured by an oralglucose tolerance test (2 g/kg glucose) after two weeks of treatmentwith either mAb22810 NPR1 agonist mAb, hFc.FGF21 or an isotype controlmAb. FIG. 20B shows glucose levels following an overnight fast asrecorded at the start of the study in A. After two weeks of treatment,mice from each group were fasted overnight in clean cages and given anoral glucose load of 2 g/kg the following morning. Glucose was thenrecorded by tail vein using a hand-held glucometer at T0, T15, T30, T60,T90 and T120. A human IgG4 antibody was used as isotype control. Allvalues are mean±SEM, n=10 per group. hFc.FGF21 group: **=P<0.01 vsisotype control, ***=P<0.001 vs isotype control, ****=P<0.0001 vsisotype control. mAb22810 group: ++=P<0.01 vs isotype control.Statistics by two-way ANOVA+Bonferonni for the oGTT, one-wayANOVA+Bonferonni for the fasting glucose.

FIG. 21A shows changes in body weight following administration ofmAb22033 NPR1 agonist mAb, hFc.FGF21 or an isotype control mAb. FIG. 21Band FIG. 21C show total fat and total lean mass respectively after sixweeks of treatment as measured by EchoMRI. NPR1 humanized mice were madeobese by placing them on a 60% high-fat diet for 10 weeks. Followingthis period, mice were randomized into three groups of equal body weightand given a subcutaneous injection of treatments at the doses andfrequencies listed in Table 45. A human IgG4 antibody was used asisotype control. All values are mean±SEM, n=10 per group. *=P<0.05 vsisotype control, ****=P<0.0001 vs isotype control. Statistics by two-wayANOVA+Tukey for the body weights, one-way ANOVA+Bonferonni for the fatand lean mass.

FIGS. 22A, 22B, and 22C show changes in VO₂ (FIG. 22A), VCO₂ (FIG. 22B)or Energy Expenditure (FIG. 22C) broken down as the average of eachday/night cycle after one week of treatment with either mAb22033 NPR1agonist mAb, hFc.FGF21 or an isotype control mAb. After one week oftreatment, mice from each group were placed in a Columbia Instrumentsmetabolic cage system (CLAMS) for one week to record metabolicparameters. Mice were acclimated to the cages for one week prior tomeasurement to minimize stress. A human IgG4 antibody was used asisotype control. All values are mean±SEM, n=5-6 per group. ****=P<0.0001vs isotype control. Statistics by one-way ANOVA+Bonferonni.

FIGS. 23A and 23B show the effects of anti-NPR1 antibody mAb22033 onglucose tolerance, as shown by blood glucose (FIG. 23A) and fastingglucose (FIG. 23B) levels. After four weeks of treatment, mice from eachgroup were fasted overnight in clean cages and given an oral glucoseload of 2 g/kg the following morning. Glucose was then recorded by tailvein using a hand-held glucometer at T0, T15, T30, T60, T90 and T120. Ahuman IgG4 antibody was used as isotype control. All values aremean±SEM, n=10 per group. hFc.FGF21 group: ***=P<0.001 vs isotypecontrol, ****=P<0.0001 vs isotype control. mAb22033 group: +=P<0.05 vsisotype control, ++=P<0.01 vs isotype control. Statistics by two-wayANOVA+Bonferonni for the oGTT, one-way ANOVA+Bonferonni for the fastingglucose.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Definitions

The term “NPR1”, also called “NPRA” refers to natriuretic peptidereceptor 1 (also known as natriuretic peptide receptor A). NPR1 is ahomodimeric transmembrane guanylate cyclase, an enzyme that catalyzescGMP synthesis. NPR1 is the receptor for both atrial (ANP) and brain(BNP) natriuretic peptides and undergoes conformational changes in theextracellular domain upon ligand binding (Ogawa et al 2004, J. Biol.Chem. 279: 28625-31). The protein has 4 distinct regions comprising anextracellular ligand-binding domain, a single transmembrane-spanningregion, an intracellular protein kinase-like homology domain and aguanylyl cyclase catalytic domain. The amino acid sequence offull-length NPR1 protein is exemplified by the amino acid sequenceprovided in UniProtKB/Swiss-Prot as accession number P16066.1 (SEQ IDNO: 193). The term “NPR1” includes recombinant NPR1 protein or afragment thereof. The term also encompasses NPR1 protein or a fragmentthereof coupled to, for example, histidine tag, mouse or human Fc, or asignal sequence such as ROR1 (for example, SEQ ID NOs: 194-199).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds(i.e., “full antibody molecules”), as well as multimers thereof (e.g.IgM) or antigen-binding fragments thereof. Each heavy chain is comprisedof a heavy chain variable region (“HCVR” or “V_(H)”) and a heavy chainconstant region (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Eachlight chain is comprised of a light chain variable region (“LCVR or“V_(L)”) and a light chain constant region (C_(L)). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, theFRs of the antibody (or antigen-binding fragment thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The fully human anti-NPR1 monoclonal antibodies disclosed herein maycomprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. The presentinvention includes antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments thatcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic biological properties, reduced immunogenicity, etc.Antibodies and antigen-binding fragments obtained in this general mannerare encompassed within the present invention.

The present invention also includes fully human anti-NPR1 monoclonalantibodies comprising variants of any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includes anti-NPR1antibodies having HCVR, LCVR, and/or CDR amino acid sequences with,e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservativeamino acid substitutions relative to any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein.

The term “human antibody”, or “fully human antibody”, as used herein, isintended to include antibodies having variable and constant regionsderived from human germline immunoglobulin sequences. The human mAbs ofthe invention may include amino acid residues not encoded by humangermline immunoglobulin sequences (e.g., mutations introduced by randomor site-specific mutagenesis in vitro or by somatic mutation in vivo),for example in the CDRs and in particular CDR3. However, the term “humanantibody”, or “fully human antibody”, as used herein, is not intended toinclude mAbs in which CDR sequences derived from the germline of anothermammalian species (e.g., mouse), have been grafted onto human FRsequences. The term includes antibodies that are recombinantly producedin a non-human mammal, or in cells of a non-human mammal. The term isnot intended to include antibodies isolated from or generated in a humansubject.

The term “recombinant”, as used herein, refers to antibodies orantigen-binding fragments thereof of the invention created, expressed,isolated or obtained by technologies or methods known in the art asrecombinant DNA technology which include, e.g., DNA splicing andtransgenic expression. The term refers to antibodies expressed in anon-human mammal (including transgenic non-human mammals, e.g.,transgenic mice), or a cell (e.g., CHO cells) expression system orisolated from a recombinant combinatorial human antibody library.

The term “specifically binds,” or “binds specifically to”, or the like,means that an antibody or antigen-binding fragment thereof forms acomplex with an antigen that is relatively stable under physiologicconditions. Specific binding can be characterized by an equilibriumdissociation constant of at least about 1×10⁻⁸ M or less (e.g., asmaller K_(D) denotes a tighter binding). Methods for determiningwhether two molecules specifically bind are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. As described herein, antibodies have been identified bysurface plasmon resonance, e.g., BIACORE™, which bind specifically toNPR1. Moreover, multi-specific antibodies that bind to one domain inNPR1 and one or more additional antigens or a bi-specific that binds totwo different regions of NPR1 are nonetheless considered antibodies that“specifically bind”, as used herein.

The term “high affinity” antibody refers to those mAbs having a bindingaffinity to NPR1, expressed as K_(D), of at least 10⁻⁸ M; preferably10⁻⁹ M; more preferably 10⁻¹⁰M, even more preferably 10-11 M, asmeasured by surface plasmon resonance, e.g., BIACORE™ orsolution-affinity ELISA.

By the term “slow off rate”, “Koff” or “kd” is meant an antibody thatdissociates from NPR1, with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴ s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

The terms “antigen-binding portion” of an antibody, “antigen-bindingfragment” of an antibody, and the like, as used herein, include anynaturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. The terms “antigen-binding fragment” of anantibody, or “antibody fragment”, as used herein, refers to one or morefragments of an antibody that retain the ability to bind to NPR1protein.

In specific embodiments, antibody or antibody fragments of the inventionmay be conjugated to a moiety such a ligand or a therapeutic moiety(“immunoconjugate”), a second anti-NPR1 antibody, or any othertherapeutic moiety useful for treating a NPR1-associated disease ordisorder.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies (Abs) havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds NPR1, or a fragment thereof, is substantially free ofAbs that specifically bind antigens other than NPR1.

An “activating antibody” or an “agonist antibody”, as used herein (or an“antibody that increases or potentiates NPR1 activity” or “an antibodythat stabilizes the activated conformation”), is intended to refer to anantibody whose binding to NPR1 results in activation of at least onebiological activity of NPR1. For example, an antibody of the inventionmay decrease systemic blood pressure upon administration to a subject inneed thereof.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timebiomolecular interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIACORE™system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. The term“epitope” also refers to a site on an antigen to which B and/or T cellsrespond. It also refers to a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The term “cross-competes”, as used herein, means an antibody orantigen-binding fragment thereof binds to an antigen and inhibits orblocks the binding of another antibody or antigen-binding fragmentthereof. The term also includes competition between two antibodies inboth orientations, i.e., a first antibody that binds and blocks bindingof second antibody and vice-versa. In certain embodiments, the firstantibody and second antibody may bind to the same epitope.Alternatively, the first and second antibodies may bind to different,but overlapping epitopes such that binding of one inhibits or blocks thebinding of the second antibody, e.g., via steric hindrance.Cross-competition between antibodies may be measured by methods known inthe art, for example, by a real-time, label-free bio-layerinterferometry assay. Cross-competition between two antibodies may beexpressed as the binding of the second antibody that is less than thebackground signal due to self-self binding (wherein first and secondantibodies is the same antibody). Cross-competition between 2 antibodiesmay be expressed, for example, as % binding of the second antibody thatis less than the baseline self-self background binding (wherein firstand second antibodies is the same antibody).

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below. A nucleic acid molecule havingsubstantial identity to a reference nucleic acid molecule may, incertain instances, encode a polypeptide having the same or substantiallysimilar amino acid sequence as the polypeptide encoded by the referencenucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positions,which are not identical, differ by conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment are wellknown to those of skill in the art. See, e.g., Pearson (1994) MethodsMol. Biol. 24: 307-331, which is herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT, which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997)Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated byreference.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

As used herein, the term “subject” refers to an animal, preferably amammal, more preferably a human, in need of amelioration, preventionand/or treatment of a NPR1-associated disease or disorder such ashypertension. The term includes human subjects who have or are at riskof having such a disease or disorder.

As used herein, the terms “treat”, “treating”, or “treatment” refer tothe reduction or amelioration of the severity of at least one symptom orindication of a NPR1-associated disease or disorder due to theadministration of a therapeutic agent such as an antibody of the presentinvention to a subject in need thereof. The terms include inhibition ofprogression of disease or of worsening of a symptom/indication. Theterms also include positive prognosis of disease, i.e., the subject maybe free of disease or may have reduced disease upon administration of atherapeutic agent such as an antibody of the present invention. Thetherapeutic agent may be administered at a therapeutic dose to thesubject.

The terms “prevent”, “preventing” or “prevention” refer to inhibition ofmanifestation of a NPR1-associated disease or disorder or any symptomsor indications of such a disease or disorder upon administration of anantibody of the present invention.

Antigen-Binding Fragments of Antibodies

Unless specifically indicated otherwise, the term “antibody,” as usedherein, shall be understood to encompass antibody molecules comprisingtwo immunoglobulin heavy chains and two immunoglobulin light chains(i.e., “full antibody molecules”) as well as antigen-binding fragmentsthereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. The terms “antigen-binding fragment”of an antibody, or “antibody fragment”, as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to NPR1 protein. An antibody fragment may include a Fab fragment, aF(ab′)₂ fragment, a Fv fragment, a dAb fragment, a fragment containing aCDR, or an isolated CDR. In certain embodiments, the term“antigen-binding fragment” refers to a polypeptide fragment of amulti-specific antigen-binding molecule. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and (optionally)constant domains. Such DNA is known and/or is readily available from,e.g., commercial sources, DNA libraries (including, e.g., phage-antibodylibraries), or can be synthesized. The DNA may be sequenced andmanipulated chemically or by using molecular biology techniques, forexample, to arrange one or more variable and/or constant domains into asuitable configuration, or to introduce codons, create cysteineresidues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (V)V_(H)-C_(H)1-C_(H)2-C_(H)3; V_(H)-C_(H)2-C_(H)3; (Vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemono-specific or multi-specific (e.g., bi-specific). A multi-specificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multi-specific antibody format, including theexemplary bi-specific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind toNPR1.

An immunogen comprising any one of the following can be used to generateantibodies to NPR1 protein. In certain embodiments, the antibodies ofthe invention are obtained from mice immunized with a full length,native NPR1 protein (See, for example, UniProtKB/Swiss-Prot accessionnumber P16066.1) or with DNA encoding the protein or fragment thereof.Alternatively, the protein or a fragment thereof may be produced usingstandard biochemical techniques and modified and used as immunogen.

In some embodiments, the immunogen may be a recombinant NPR1 protein orfragment thereof expressed in E. coli or in any other eukaryotic ormammalian cells such as Chinese hamster ovary (CHO) cells (for example,SEQ ID NOs: 194-199)

Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other knownmethod for generating monoclonal antibodies, high affinity chimericantibodies to NPR1 are initially isolated having a human variable regionand a mouse constant region. The VELOCIMMUNE® technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are replaced with a desired humanconstant region to generate the fully human antibody of the invention,for example wild-type or modified IgG1 or IgG4. While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

Bioequivalents

The anti-NPR1 antibodies and antibody fragments of the present inventionencompass proteins having amino acid sequences that vary from those ofthe described antibodies, but that retain the ability to bind NPR1protein. Such variant antibodies and antibody fragments comprise one ormore additions, deletions, or substitutions of amino acids when comparedto parent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, theantibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an antibody or antibody fragment that is essentiallybioequivalent to an antibody or antibody fragment of the invention.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single dose or multipledoses. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,or potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of the antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include antibody variants comprising aminoacid changes, which modify the glycosylation characteristics of theantibodies, e.g., mutations that eliminate or remove glycosylation.

Anti-NPR1 Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-NPR1antibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-NPR1 antibodies comprising a mutation inthe C_(H)2 or a C_(H)3 region of the Fc domain, wherein the mutation(s)increases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Suchmutations may result in an increase in serum half-life of the antibodywhen administered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position 250 (e.g., E orQ); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W, H, F or Y[N434A, N434W, N434H, N434F or N434Y]); or a modification at position250 and/or 428; or a modification at position 307 or 308 (e.g., 308F,V308F), and 434. In one embodiment, the modification comprises a 428L(e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g.,V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T,and 256E) modification; a 250Q and 428L modification (e.g., T250Q andM428L); and a 307 and/or 308 modification (e.g., 308F or 308P). In yetanother embodiment, the modification comprises a 265A (e.g., D265A)and/or a 297A (e.g., N297A) modification.

For example, the present invention includes anti-NPR1 antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I andQ311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g.,D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A);and 433K and 434F (e.g., H433K and N434F). All possible combinations ofthe foregoing Fc domain mutations and other mutations within theantibody variable domains disclosed herein, are contemplated within thescope of the present invention.

The present invention also includes anti-NPR1 antibodies comprising achimeric heavy chain constant (C_(H)) region, wherein the chimeric C_(H)region comprises segments derived from the C_(H) regions of more thanone immunoglobulin isotype. For example, the antibodies of the inventionmay comprise a chimeric C_(H) region comprising part or all of a C_(H)2domain derived from a human IgG1, human IgG2 or human IgG4 molecule,combined with part or all of a C_(H)3 domain derived from a human IgG1,human IgG2 or human IgG4 molecule. According to certain embodiments, theantibodies of the invention comprise a chimeric C_(H) region having achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” amino acid sequence (amino acid residues from positions216 to 227 according to EU numbering) derived from a human IgG1, a humanIgG2 or a human IgG4 hinge region, combined with a “lower hinge”sequence (amino acid residues from positions 228 to 236 according to EUnumbering) derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion. According to certain embodiments, the chimeric hinge regioncomprises amino acid residues derived from a human IgG1 or a human IgG4upper hinge and amino acid residues derived from a human IgG2 lowerhinge. An antibody comprising a chimeric C_(H) region as describedherein may, in certain embodiments, exhibit modified Fc effectorfunctions without adversely affecting the therapeutic or pharmacokineticproperties of the antibody. (See, e.g., U.S. Patent ApplicationPublication 2014/0243504, the disclosure of which is hereby incorporatedby reference in its entirety).

Biological Characteristics of the Antibodies

In general, the antibodies of the present invention function by bindingto NPR1 protein and increasing its activity. For example, the presentinvention includes antibodies and antigen-binding fragments ofantibodies that bind monomeric human NPR1 protein in the absence ofeither ANP or BNP (e.g., at 25° C. or at 37° C.) with a K_(D) of lessthan 690 nM as measured by surface plasmon resonance, e.g., using theassay format as defined in Example 3 herein. In certain embodiments, theantibodies or antigen-binding fragments thereof bind NPR1 with a K_(D)of less than about 650 nM, less than about 500 nM, less than about 400nM, less than about 300 nM, less than about 200 nM, less than about 100nM, less than about 50 nM, or less than about 25 nM, as measured bysurface plasmon resonance, e.g., using the assay format as defined inExample 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind dimeric human NPR1 protein in theabsence of either ANP or BNP (e.g., at 25° C. or at 37° C.) with a K_(D)of less than 42 nM as measured by surface plasmon resonance, e.g., usingthe assay format as defined in Example 3 herein. In certain embodiments,the antibodies or antigen-binding fragments thereof bind NPR1 with aK_(D) of less than about 40 nM, less than about 30 nM, less than about20 nM, less than about 10 nM, less than about 5 nM, less than about 1nM, or less than about 0.5 nM, as measured by surface plasmon resonance,e.g., using the assay format as defined in Example 3 herein, or asubstantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind human NPR1 protein complexed to ANP(e.g., at 25° C. or at 37° C.) with a K_(D) of less than 80 nM asmeasured by surface plasmon resonance, e.g., using the assay format asdefined in Example 3 herein. In certain embodiments, the antibodies orantigen-binding fragments thereof bind NPR1 with a K_(D) of less thanabout 70 nM, less than about 50 nM, less than about 25 nM, less thanabout 10 nM, less than about 5 nM, less than about 1 nM, or less thanabout 0.5 nM, as measured by surface plasmon resonance, e.g., using theassay format as defined in Example 3 herein, or a substantially similarassay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind human NPR1 protein complexed to BNP(e.g., at 25° C. or at 37° C.) with a K_(D) of less than 20 nM asmeasured by surface plasmon resonance, e.g., using the assay format asdefined in Example 3 herein. In certain embodiments, the antibodies orantigen-binding fragments thereof bind NPR1 with a K_(D) of less thanabout 15 nM, less than about 10 nM, less than about 5 nM, less thanabout 1 nM, or less than about 0.5 nM, as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein,or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind monomeric monkey NPR1 protein in theabsence of either ANP or BNP (e.g., at 25° C. or at 37° C.) with a K_(D)of less than 365 nM as measured by surface plasmon resonance, e.g.,using the assay format as defined in Example 3 herein. In certainembodiments, the antibodies or antigen-binding fragments thereof bindNPR1 with a K_(D) of less than about 360 nM, less than about 300 nM,less than about 150 nM, less than about 100 nM, less than about 50 nM,less than about 25 nM, less than about 10 nM, less than about 5 nM, lessthan about 1 nM, or less than about 0.5 nM, as measured by surfaceplasmon resonance, e.g., using the assay format as defined in Example 3herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind dimeric monkey NPR1 protein in theabsence of either ANP or BNP (e.g., at 25° C. or at 37° C.) with a K_(D)of less than 30 nM as measured by surface plasmon resonance, e.g., usingthe assay format as defined in Example 3 herein. In certain embodiments,the antibodies or antigen-binding fragments thereof bind NPR1 with aK_(D) of less than about 20 nM, less than about 10 nM, less than about 5nM, less than about 1 nM, or less than about 0.5 nM, as measured bysurface plasmon resonance, e.g., using the assay format as defined inExample 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind monkey NPR1 protein complexed to ANP(e.g., at 25° C. or at 37° C.) with a K_(D) of less than 10 nM asmeasured by surface plasmon resonance, e.g., using the assay format asdefined in Example 3 herein. In certain embodiments, the antibodies orantigen-binding fragments thereof bind NPR1 with a K_(D) of less thanabout 9 nM, less than about 8 nM, less than about 5 nM, less than about1 nM, or less than about 0.5 nM, as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein,or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind dimeric monkey NPR1 protein complexedto BNP (e.g., at 25° C. or at 37° C.) with a K_(D) of less than 10 nM asmeasured by surface plasmon resonance, e.g., using the assay format asdefined in Example 3 herein. In certain embodiments, the antibodies orantigen-binding fragments thereof bind NPR1 with a K_(D) of less thanabout 9 nM, less than about 8 nM, less than about 5 nM, less than about1 nM, or less than about 0.5 nM, as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein,or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind to cells expressing human NPR1 with orwithout ANP at a EC₅₀ of less than 5 nM, less than 4 nM, less than 3 nM,less than 2 nM, less than 1 nM, or less than 0.5 nM, as measured, e.g.,using an assay format as described in Example 5 herein, or asubstantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that activate NPR1 with a EC₅₀ of less than 385 nM, asmeasured by a calcium flux cell-based bioassay, e.g., using the assayformat as defined in Example 6 herein. In certain embodiments, theantibodies or antigen-binding fragments thereof activate NPR1 with aEC₅₀ of less than about 300 nM, less than about 200 nM, less than about100 nM, less than about 50 nM, less than about 10 nM, or less than about1 nM, as measured by calcium flux cell-based bioassay, e.g., using theassay format as defined in Example 6 herein, or a substantially similarassay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind to NPR1 and decrease the systemicblood pressure of a subject for more than 28 days when administered tothe subject in need thereof as a single dose, e.g., as shown in Example8 herein.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind to NPR1 and reduced fasting bloodglucose levels when administered to a subject in need thereof, e.g., asshown in Example 11 herein.

In one embodiment, the present invention provides an isolatedrecombinant antibody or antigen-binding fragment thereof that bindsspecifically to NPR1 protein in the presence or absence of ANP or BNPand increases the activity of NPR1, wherein the antibody or fragmentthereof exhibits one or more of the following characteristics: (a) is afully human monoclonal antibody; (b) binds to monomeric human NPR1 inthe absence of ANP and/or BNP at 25° C. and at 37° C. with adissociation constant (K_(D)) of less than 690 nM, as measured in asurface plasmon resonance assay; (c) binds to dimeric human NPR1 in theabsence of ANP or BNP at 25° C. and at 37° C. with a K_(D) of less than42 nM, as measured in a surface plasmon resonance assay; (d) binds tohuman NPR1 complexed to ANP at 25° C. and 37° C. with a K_(D) of lessthan 80 nM, as measured in a surface plasmon resonance assay; (e) bindsto human NPR1 complexed to BNP at 25° C. and 37° C. with a K_(D) of lessthan 20 nM, as measured in a surface plasmon resonance assay; (f) bindsto monomeric monkey NPR1 in the absence of ANP and/or BNP at 25° C. and37° C. with a K_(D) of less than 365 nM, as measured in a surfaceplasmon resonance assay; (g) binds to dimeric monkey NPR1 in the absenceof ANP or BNP at 25° C. and at 37° C. with a K_(D) of less than 30 nM,as measured in a surface plasmon resonance assay; (h) binds to monkeyNPR1 complexed to ANP at 25° C. and 37° C. with a K_(D) of less than 10nM, as measured in a surface plasmon resonance assay; (i) binds tomonkey NPR1 complexed to BNP at 25° C. and 37° C. with a K_(D) of lessthan 10 nM, as measured in a surface plasmon resonance assay; (j) doesnot bind to mouse NPR1; (k) binds to cells expressing human NPR1 (in theabsence of ANP) or NPR1-complexed to ANP with EC₅₀ less than 5 nM; (l)activates NPR1 with a EC₅₀ of less than 385 nM, as measured in a calciumflux cell-based bioassay; (m) reduces the systemic blood pressure whenadministered to normotensive and hypertensive mice, wherein thereduction in systemic and mean arterial blood pressures lasts for up to28 days upon administration of a single dose; (n) improves glucosetolerance when administered to diet-induced obese mice; and (o)comprises a HCVR comprising an amino acid sequence selected from thegroup consisting of HCVR sequence listed in Table 1 and a LCVRcomprising an amino acid sequence selected from the group consisting ofLCVR sequences listed in Table 1.

The antibodies of the present invention may possess one or more of theaforementioned biological characteristics, or any combinations thereof.Other biological characteristics of the antibodies of the presentinvention will be evident to a person of ordinary skill in the art froma review of the present disclosure including the working Examplesherein.

Epitope Mapping and Related Technologies

The present invention includes anti-NPR1 antibodies that interact withone or more amino acids found within one or more regions of the NPR1protein molecule. The epitope to which the antibodies bind may consistof a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids locatedwithin any of the aforementioned domains of the NPR1 protein molecule(e.g. a linear epitope in a domain). Alternatively, the epitope mayconsist of a plurality of non-contiguous amino acids (or amino acidsequences) located within either or both of the aforementioned domainsof the protein molecule (e.g. a conformational epitope).

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,for example, routine cross-blocking assays, such as that described inAntibodies, Harlow and Lane (Cold Spring Harbor Press, Cold SpringHarbor, N.Y.). Other methods include alanine scanning mutationalanalysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis crystallographic studies and NMRanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Prot. Sci. 9: 487-496). Another method that can be used toidentify the amino acids within a polypeptide with which an antibodyinteracts is hydrogen/deuterium exchange detected by mass spectrometry.In general terms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water and exchangeable protons within aminoacids that are protected by the antibody complex undergodeuterium-to-hydrogen back-exchange at a slower rate than exchangeableprotons within amino acids that are not part of the interface. As aresult, amino acids that form part of the protein/antibody interface mayretain deuterium and therefore exhibit relatively higher mass comparedto amino acids not included in the interface. After dissociation of theantibody, the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residuesthat correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (seeUS 2004/0101920, herein specifically incorporated by reference in itsentirety). Each category may reflect a unique epitope either distinctlydifferent from or partially overlapping with epitope represented byanother category. This technology allows rapid filtering of geneticallyidentical antibodies, such that characterization can be focused ongenetically distinct antibodies. When applied to hybridoma screening,MAP may facilitate identification of rare hybridoma clones that producemAbs having the desired characteristics. MAP may be used to sort theantibodies of the invention into groups of antibodies binding differentepitopes.

In certain embodiments, the present invention includes anti-NPR1antibodies and antigen-binding fragments thereof that interact with oneor more epitopes found within the extracellular domain of NPR1. Theepitope(s) may consist of one or more contiguous sequences of 3 or more(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20or more) amino acids located within the extracellular domain of NPR1.Alternatively, the epitope may consist of a plurality of non-contiguousamino acids (or amino acid sequences) located within NPR1.

The present invention includes anti-NPR1 antibodies that bind to thesame epitope, or a portion of the epitope, as any of the specificexemplary antibodies listed in Table 1. Likewise, the present inventionalso includes anti-NPR1 antibodies that compete for binding to NPR1protein or a fragment thereof with any of the specific exemplaryantibodies listed in Table 1. For example, the present inventionincludes anti-NPR1 antibodies that cross-compete for binding to NPR1protein with one or more antibodies listed in Table 1.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-NPR1 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-NPR1antibody of the invention, the reference antibody is allowed to bind toa NPR1 protein or peptide under saturating conditions. Next, the abilityof a test antibody to bind to the NPR1 protein molecule is assessed. Ifthe test antibody is able to bind to NPR1 following saturation bindingwith the reference anti-NPR1 antibody, it can be concluded that the testantibody binds to a different epitope than the reference anti-NPR1antibody. On the other hand, if the test antibody is not able to bind tothe NPR1 protein following saturation binding with the referenceanti-NPR1 antibody, then the test antibody may bind to the same epitopeas the epitope bound by the reference anti-NPR1 antibody of theinvention.

To determine if an antibody competes for binding with a referenceanti-NPR1 antibody, the above-described binding methodology is performedin two orientations: In a first orientation, the reference antibody isallowed to bind to a NPR1 protein under saturating conditions followedby assessment of binding of the test antibody to the NPR1 molecule. In asecond orientation, the test antibody is allowed to bind to a NPR1molecule under saturating conditions followed by assessment of bindingof the reference antibody to the NPR1 molecule. If, in bothorientations, only the first (saturating) antibody is capable of bindingto the NPR1 molecule, then it is concluded that the test antibody andthe reference antibody compete for binding to NPR1. As will beappreciated by a person of ordinary skill in the art, an antibody thatcompetes for binding with a reference antibody may not necessarily bindto the identical epitope as the reference antibody, but may stericallyblock binding of the reference antibody by binding an overlapping oradjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have thesame epitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

In certain embodiments, the present invention provides an isolatedantibody or antigen-binding fragment thereof that binds specifically tonatriuretic peptide receptor 1 (NPR1) protein, wherein the antibody orantigen-binding fragment thereof interacts with one or more amino acidscontained within the extracellular domain of NPR1 (amino acids 29-347 ofSEQ ID NO: 194), as determined by hydrogen/deuterium exchange, andwherein the antibody or antigen-binding fragment thereof: (i) binds tocells expressing human NPR1 in the presence or absence of atrialnatriuretic peptide (ANP); and/or (ii) binds to NPR1 and activates NPR1.In one embodiment, the antibody or antigen-binding fragment thereofinteracts with an amino acid sequence selected from the group consistingof (a) amino acids 29 to 45 of SEQ ID NO: 194; (b) amino acids 331 to347 of SEQ ID NO: 194; (c) amino acids 336 to 347 of SEQ ID NO: 194; (d)amino acids 331 to 335 of SEQ ID NO: 194; and (e) amino acids 70 to 81of SEQ ID NO: 194. In one embodiment, the antibody or antigen-bindingfragment thereof interacts with an amino acid sequence selected from thegroup consisting of (a) amino acids 29 to 45 of SEQ ID NO: 194; and (b)amino acids 336 to 347 of SEQ ID No: 194. In one embodiment, theantibody or antigen-binding fragment thereof interacts with an aminoacid sequence selected from the group consisting of (a) amino acids 29to 45 of SEQ ID NO: 194; and (b) amino acids 331 to 347 of SEQ ID No:194, in the presence of ANP. In one embodiment, the antibody orantigen-binding fragment thereof interacts with an amino acid sequenceselected from the group consisting of (a) amino acids 29 to 45 of SEQ IDNO: 194; (b) amino acids 70 to 81 of SEQ ID NO: 194; and (c) amino acids331 to 335 of SEQ ID No: 194, in the presence of ANP. In one embodiment,the present invention provides an isolated antibody or antigen-bindingfragment thereof that binds specifically to NPR1 protein in the presenceof ANP, wherein the antibody or antigen-binding fragment thereofinteracts with an amino acid sequence selected from the group consistingof (a) amino acids 29 to 45 of SEQ ID NO: 194; and (b) amino acids 331to 347 of SEQ ID No: 194, but not with amino acids 70 to 81 of SEQ IDNO: 194, as determined by hydrogen/deuterium exchange, and wherein theantibody or antigen-binding fragment thereof: (i) binds to cellsexpressing human NPR1 in the presence or absence of ANP; and/or (ii)binds to NPR1 and activates NPR1.

Immunoconjugates

The invention encompasses a human anti-NPR1 monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), to treat aNPR1-associated disease or disorder (e.g., hypertension). As usedherein, the term “immunoconjugate” refers to an antibody that ischemically or biologically linked to a radioactive agent, a cytokine, aninterferon, a target or reporter moiety, an enzyme, a peptide or proteinor a therapeutic agent. The antibody may be linked to the radioactiveagent, cytokine, interferon, target or reporter moiety, enzyme, peptideor therapeutic agent at any location along the molecule so long as it isable to bind its target. Examples of immunoconjugates include antibodydrug conjugates and antibody-toxin fusion proteins. In one embodiment,the agent may be a second different antibody to NPR1 protein. The typeof therapeutic moiety that may be conjugated to the anti-NPR1 antibodyand will take into account the condition to be treated and the desiredtherapeutic effect to be achieved. Examples of suitable agents forforming immunoconjugates are known in the art; see for example, WO05/103081.

Multi-Specific Antibodies

The antibodies of the present invention may be mono-specific,bi-specific, or multi-specific. Multi-specific antibodies may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244.

Any of the multi-specific antigen-binding molecules of the invention, orvariants thereof, may be constructed using standard molecular biologicaltechniques (e.g., recombinant DNA and protein expression technology), aswill be known to a person of ordinary skill in the art.

In some embodiments, NPR1-specific antibodies are generated in abi-specific format (a “bi-specific”) in which variable regions bindingto distinct domains of NPR1 protein are linked together to conferdual-domain specificity within a single binding molecule. Appropriatelydesigned bi-specifics may enhance overall NPR1-protein inhibitoryefficacy through increasing both specificity and binding avidity.Variable regions with specificity for individual domains, (e.g.,segments of the N-terminal domain), or that can bind to differentregions within one domain, are paired on a structural scaffold thatallows each region to bind simultaneously to the separate epitopes, orto different regions within one domain. In one example for abi-specific, heavy chain variable regions (V_(H)) from a binder withspecificity for one domain are recombined with light chain variableregions (V_(L)) from a series of binders with specificity for a seconddomain to identify non-cognate V_(L) partners that can be paired with anoriginal V_(H) without disrupting the original specificity for thatV_(H). In this way, a single V_(L) segment (e.g., V_(L)1) can becombined with two different V_(H) domains (e.g., V_(H)1 and V_(H)2) togenerate a bi-specific comprised of two binding “arms” (V_(H)1-V_(L)1and V_(H)2-V_(L)1). Use of a single V_(L) segment reduces the complexityof the system and thereby simplifies and increases efficiency incloning, expression, and purification processes used to generate thebi-specific (See, for example, US2011/0195454 and US2010/0331527).

Alternatively, antibodies that bind more than one domains and a secondtarget, such as, but not limited to, for example, a second differentanti-NPR1 antibody, may be prepared in a bi-specific format usingtechniques described herein, or other techniques known to those skilledin the art. Antibody variable regions binding to distinct regions may belinked together with variable regions that bind to relevant sites on,for example, the extracellular domain of NPR1, to confer dual-antigenspecificity within a single binding molecule. Appropriately designedbi-specifics of this nature serve a dual function. Variable regions withspecificity for the extracellular domain are combined with a variableregion with specificity for outside the extracellular domain and arepaired on a structural scaffold that allows each variable region to bindto the separate antigens.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bi-specific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mabe bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the anti-NPR1antibodies or antigen-binding fragments thereof of the presentinvention. Therapeutic compositions in accordance with the inventionwill be administered with suitable carriers, excipients, and otheragents that are incorporated into formulations to provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose of antibody may vary depending upon the age and the size of asubject to be administered, target disease, conditions, route ofadministration, and the like. When an antibody of the present inventionis used for treating a disease or disorder in an adult patient, or forpreventing such a disease, it is advantageous to administer the antibodyof the present invention normally at a single dose of about 0.1 to about100 mg/kg body weight. Depending on the severity of the condition, thefrequency and the duration of the treatment can be adjusted. In certainembodiments, the antibody or antigen-binding fragment thereof of theinvention can be administered as an initial dose of at least about 0.1mg to about 800 mg, about 1 to about 600 mg, about 5 to about 500 mg, orabout 10 to about 400 mg. In certain embodiments, the initial dose maybe followed by administration of a second or a plurality of subsequentdoses of the antibody or antigen-binding fragment thereof in an amountthat can be approximately the same or less than that of the initialdose, wherein the subsequent doses are separated by at least 1 day to 3days; at least one week, at least 2 weeks; at least 3 weeks; at least 4weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or atleast 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. The pharmaceutical composition can be also deliveredin a vesicle, in particular a liposome (see, for example, Langer (1990)Science 249:1527-1533).

The use of nanoparticles to deliver the antibodies of the presentinvention is also contemplated herein. Antibody-conjugated nanoparticlesmay be used both for therapeutic and diagnostic applications.Antibody-conjugated nanoparticles and methods of preparation and use aredescribed in detail by Arruebo, M., et al. 2009 (“Antibody-conjugatednanoparticles for biomedical applications” in J. Nanomat. Volume 2009,Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporatedherein by reference. Nanoparticles may be developed and conjugated toantibodies contained in pharmaceutical compositions to target cells.Nanoparticles for drug delivery have also been described in, forexample, U.S. Pat. No. 8,257,740, or U.S. Pat. No. 8,246,995, eachincorporated herein in its entirety.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracranial, intraperitoneal and intramuscularinjections, drip infusions, etc. These injectable preparations may beprepared by methods publicly known.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the antibody contained isgenerally about 5 to about 500 mg per dosage form in a unit dose;especially in the form of injection, it is preferred that the antibodyis contained in about 5 to about 300 mg and in about 10 to about 300 mgfor the other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the present invention are useful for the treatment,and/or prevention of a disease or disorder or condition associated withNPR1 and/or for ameliorating at least one symptom associated with suchdisease, disorder or condition. In certain embodiments, an antibody orantigen-binding fragment thereof of the invention may be administered ata therapeutic dose to a patient with a disease or disorder or conditionassociated with NPR1.

In certain embodiments, the antibodies of the present invention areuseful for treating or preventing at least one symptom or indication ofa NPR1-associated disease or disorder selected from the group consistingof hypertension, heart failure, obesity, renal failure, chronic kidneydisease, macular edema, glaucoma, stroke, lung disorders, pulmonaryfibrosis, inflammation, asthma, skeletal growth disorders, bonefractures, diabetes, and cancer.

It is also contemplated herein to use one or more antibodies of thepresent invention prophylactically to subjects at risk for sufferingfrom a NPR1-associated disease or disorder.

In one embodiment of the invention, the present antibodies are used forthe preparation of a pharmaceutical composition or medicament fortreating patients suffering from a disease, disorder or conditiondisclosed herein. In another embodiment of the invention, the presentantibodies are used as adjunct therapy with any other agent or any othertherapy known to those skilled in the art useful for treating orameliorating a disease, disorder or condition disclosed herein.

Combination Therapies

Combination therapies may include an antibody of the invention and anyadditional therapeutic agent that may be advantageously combined with anantibody of the invention, or with a biologically active fragment of anantibody of the invention. The antibodies of the present invention maybe combined synergistically with one or more drugs or therapy used totreat a NPR1-associated disease or disorder. In some embodiments, theantibodies of the invention may be combined with a second therapeuticagent to ameliorate one or more symptoms of said disease or condition.

Depending upon the disease, disorder or condition, the antibodies of thepresent invention may be used in combination with one or more additionaltherapeutic agents including, but not limited to, an aldosteroneantagonist (e.g., eplerenone, spironolactone), an alpha-adrenergicblocker (e.g., doxazosin, phenoxybenzamine, phentolamine, prazosin,terazosin), an angiotensin converting enzyme (ACE) inhibitor (e.g.,benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril,perindopril, quinapril, ramipril, trandolapril), an arteriolarvasodilator (e.g., hydralazine, minoxidil), an autonomic ganglionicvasodilator (e.g., mecamylamine), a beta-adrenergic blocker (acebutolol,atenolol, betaxolol, bisoprolol, carvedilol, carteolol, esmolol,labetolol, metoprolol, nadolol, penbuterol, pindolol, propranolol,timolol), a catecholamine-depleting sympatholytic (e.g., deserpidine,reserpine), a central alpha-2 adrenergic agonist (e.g., clonidine,guanabenz, guanfacine, methyldopa), a calcium channel blocker(diltiazem, verapamil, amlodipine, felodipine, isradipine, nicadipine,nifedipine, nisoldipine), a diuretic (e.g., bumetanide, ethacrynic acid,furoseamide, torsemide, chlorothiazide, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, polythiazide, chlorthalidone,indapamide, metolazone), a renin inhibitor (e.g., aliskiren), ananti-coagulant (e.g., coumardin, dabigatran, apixaban), an anti-plateletagent (e.g., aspirin, clopidogrel), a cholesterol lowering agent (e.g.,a statin, a PCSK9 inhibitor such as alirocumab), a vasodilator (e.g.,minoxidil, hydralazine, nitrates), digitalis, surgery (e.g.,angioplasty, coronary artery bypass, heart transplant), an implantabledevice (e.g., valve replacement, defibrillator device, left ventricularassist device, pacemaker), anti-tumor therapy (e.g., a chemotherapeuticagent, surgery, radiation, a PD-1 inhibitor), insulin, a GLP1 agonist(e.g., exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide,semaglutide), metformin, dialysis, bone marrow stimulant,hemofiltration, a lifestyle modification, and a dietary supplement.

As used herein, the term “in combination with” means that additionaltherapeutically active component(s) may be administered prior to,concurrent with, or after the administration of the anti-NPR1 antibodyof the present invention. The term “in combination with” also includessequential or concomitant administration of an anti-NPR1 antibody and asecond therapeutic agent.

The additional therapeutically active component(s) may be administeredto a subject prior to administration of an anti-NPR1 antibody of thepresent invention. For example, a first component may be deemed to beadministered “prior to” a second component if the first component isadministered 1 week before, 72 hours before, 60 hours before, 48 hoursbefore, 36 hours before, 24 hours before, 12 hours before, 6 hoursbefore, 5 hours before, 4 hours before, 3 hours before, 2 hours before,1 hour before, 30 minutes before, or less than 30 minutes beforeadministration of the second component. In other embodiments, theadditional therapeutically active component(s) may be administered to asubject after administration of an anti-NPR1 antibody of the presentinvention. For example, a first component may be deemed to beadministered “after” a second component if the first component isadministered 30 minutes after, 1 hour after, 2 hours after, 3 hoursafter, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24hours after, 36 hours after, 48 hours after, 60 hours after, 72 hoursafter or more after administration of the second component. In yet otherembodiments, the additional therapeutically active component(s) may beadministered to a subject concurrent with administration of an anti-NPR1antibody of the present invention. “Concurrent” administration, forpurposes of the present invention, includes, e.g., administration of ananti-NPR1 antibody and an additional therapeutically active component toa subject in a single dosage form, or in separate dosage formsadministered to the subject within about 30 minutes or less of eachother. If administered in separate dosage forms, each dosage form may beadministered via the same route (e.g., both the anti-NPR1 antibody andthe additional therapeutically active component may be administeredintravenously, etc.); alternatively, each dosage form may beadministered via a different route (e.g., the anti-NPR1 antibody may beadministered intravenously, and the additional therapeutically activecomponent may be administered orally). In any event, administering thecomponents in a single dosage from, in separate dosage forms by the sameroute, or in separate dosage forms by different routes are allconsidered “concurrent administration,” for purposes of the presentdisclosure. For purposes of the present disclosure, administration of ananti-NPR1 antibody “prior to”, “concurrent with,” or “after” (as thoseterms are defined herein above) administration of an additionaltherapeutically active component is considered administration of ananti-NPR1 antibody “in combination with” an additional therapeuticallyactive component.

The present invention includes pharmaceutical compositions in which ananti-NPR1 antibody of the present invention is co-formulated with one ormore of the additional therapeutically active component(s) as describedelsewhere herein.

Diagnostic Uses of the Antibodies

The antibodies of the present invention may be used to detect and/ormeasure NPR1 in a sample, e.g., for diagnostic purposes. Someembodiments contemplate the use of one or more antibodies of the presentinvention in assays to detect a NPR1-associated-disease or disorder.Exemplary diagnostic assays for NPR1 may comprise, e.g., contacting asample, obtained from a patient, with an anti-NPR1 antibody of theinvention, wherein the anti-NPR1 antibody is labeled with a detectablelabel or reporter molecule or used as a capture ligand to selectivelyisolate NPR1 from patient samples. Alternatively, an unlabeled anti-NPR1antibody can be used in diagnostic applications in combination with asecondary antibody which is itself detectably labeled. The detectablelabel or reporter molecule can be a radioisotope, such as ³H, ¹⁴C, ³²p,³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such asfluorescein isothiocyanate, or rhodamine; or an enzyme such as alkalinephosphatase, β-galactosidase, horseradish peroxidase, or luciferase.Specific exemplary assays that can be used to detect or measure NPR1 ina sample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in NPR1 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient, which contains detectable quantities of either NPR1 protein, orfragments thereof, under normal or pathological conditions. Generally,levels of NPR1 protein in a particular sample obtained from a healthypatient (e.g., a patient not afflicted with a disease associated withNPR1) will be measured to initially establish a baseline, or standard,level of NPR1. This baseline level of NPR1 can then be compared againstthe levels of NPR1 measured in samples obtained from individualssuspected of having a NPR1-associated condition, or symptoms associatedwith such condition.

The antibodies specific for NPR1 protein may contain no additionallabels or moieties, or they may contain an N-terminal or C-terminallabel or moiety. In one embodiment, the label or moiety is biotin. In abinding assay, the location of a label (if any) may determine theorientation of the peptide relative to the surface upon which thepeptide is bound. For example, if a surface is coated with avidin, apeptide containing an N-terminal biotin will be oriented such that theC-terminal portion of the peptide will be distal to the surface.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, room temperatureis about 25° C., and pressure is at or near atmospheric.

Example 1: Generation of Human Antibodies to Natriuretic PeptideReceptor 1 (NPR1)

Human antibodies to NPR1 protein were generated in a VELOCIMMUNE® mousecomprising DNA encoding human Immunoglobulin heavy and kappa light chainvariable regions. The mice were immunized with human NPR1 and mouse ANPDNA by hydrodynamic DNA delivery and boosted by extracellular domain ofhuman NPR1 protein complexed to mouse ANP.

The antibody immune response was monitored by a NPR1-specificimmunoassay. When a desired immune response was achieved, splenocyteswere harvested and fused with mouse myeloma cells to preserve theirviability and form hybridoma cell lines. The hybridoma cell lines werescreened and selected to identify cell lines that produce NPR1-specificantibodies. The cell lines were used to obtain several anti-NPR1chimeric antibodies (i.e., antibodies possessing human variable domainsand mouse constant domains).

Anti-NPR1 antibodies were also isolated directly from antigen-positivemouse B cells without fusion to myeloma cells, as described in U.S. Pat.No. 7,582,298, herein specifically incorporated by reference in itsentirety. Using this method, several fully human anti-NPR1 antibodies(i.e., antibodies possessing human variable domains and human constantdomains) were obtained.

Exemplary antibodies generated as disclosed above were designated asmAb22033, mAb22035, mAb22805, mAb22809, mAb22810, mAb25479, mAb25491,mAb25497, mAb25498, mAb25502, mAb25508, and mAb25545.

The biological properties of the exemplary antibodies generated inaccordance with the methods of this Example are described in detail inthe Examples set forth below.

Example 2: Heavy and Light Chain Variable Region Amino Acid andNucleotide Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-NPR1 antibodiesof the invention.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 mAb22033 2 4 6 8 10 12 1416 mAb22035 18 20 22 24 26 28 30 32 mAb22805 34 36 38 40 42 44 46 48mAb22809 50 52 54 56 58 60 62 64 mAb22810 66 68 70 72 74 76 78 80mAb25479 82 84 86 88 90 92 94 96 mAb25491 98 100 102 104 106 108 110 112mAb25497 114 116 118 120 122 124 126 128 mAb25498 130 132 134 136 138140 142 144 mAb25502 146 148 150 152 154 156 158 160 mAb25508 162 164166 168 170 172 174 176 mAb25545 178 180 182 184 186 188 190 192

The corresponding nucleic acid sequence identifiers are set forth inTable 2.

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 mAb22033 1 3 57 9 11 13 15 mAb22035 17 19 21 23 25 27 29 31 mAb22805 33 35 37 39 41 4345 47 mAb22809 49 51 53 55 57 59 61 63 mAb22810 65 67 69 71 73 75 77 79mAb25479 81 83 85 87 89 91 93 95 mAb25491 97 99 101 103 105 107 109 111mAb25497 113 115 117 119 121 123 125 127 mAb25498 129 131 133 135 137139 141 143 mAb25502 145 147 149 151 153 155 157 159 mAb25508 161 163165 167 169 171 173 175 mAb25545 177 179 181 183 185 187 189 191

Antibodies referred to herein typically have fully human variableregions, but may have human or mouse constant regions. As will beappreciated by a person of ordinary skill in the art, an antibody havinga particular Fc isotype can be converted to an antibody with a differentFc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted toan antibody with a human IgG4, etc.), but in any event, the variabledomains (including the CDRs)—which are indicated by the numericalidentifiers shown in Table 2—will remain the same, and the bindingproperties to antigen are expected to be identical or substantiallysimilar regardless of the nature of the Fc domain. In certainembodiments, selected antibodies with a mouse IgG1 Fc are converted toantibodies with human IgG4 Fc. In one embodiment, the IgG4 Fc domaincomprises 2 or more amino acid changes as disclosed in US20100331527. Inone embodiment, the human IgG4 Fc comprises a serine to proline mutationin the hinge region (S108P) to promote dimer stabilization. Unlessindicated otherwise, all antibodies used in the following examplescomprise a human IgG4 isotype.

Control Constructs Used in the Following Examples

The following control construct (anti-NPR1 antibodies) was included inthe experiments disclosed herein, for comparative purposes: “Comparator1,” a monoclonal antibody against human NPR1 having V_(H)/V_(L)sequences of antibody “mAb5591” according to US Patent No. 20120114659(Morphosys).

Example 3: Antibody Binding to NPR1 as Determined by Surface PlasmonResonance Experimental Procedure

Equilibrium dissociation constant (K_(D)) for different NPR1 reagentsbinding to purified anti-NPR1 monoclonal antibodies (mAbs) weredetermined using a real-time surface plasmon resonance based Biacore4000 biosensor. All binding studies were performed in 10 mM HEPES, 150mM NaCl, 3 mM EDTA, and 0.05% v/v Surfactant Tween-20, pH 7.4 (HBS-ET)running buffer at 25° C. and 37° C. The Biacore CM5 sensor chip surfacewas first derivatized by amine coupling with either mouse anti-human Fcspecific mAb (GE Healthcare, # BR100839) or goat anti-human Fcγ specificpolyclonal antibody (Jackson ImmunoResearch Laboratories, # BR-1008-39)to capture anti-NPR1 mAbs. Binding studies were performed on human NPR1extracellular domain expressed with a C-terminal myc-myc-hexahistidine(hNPR1-MMH) (SEQ ID NO: 194), monkey NPR1 extracellular domain expressedwith a C-terminal myc-myc-hexahistidine (mfNPR1-MMH) (SEQ ID NO: 195),mouse NPR1 extracellular domain expressed with a C-terminalmyc-myc-hexahistidine (mNPR1-MMH) (SEQ ID NO: 196), human NPR1extracellular domain expressed with a C-terminal mouse IgG2a (hNPR1-mFc)(SEQ ID NO: 197), monkey NPR1 extracellular domain expressed with aC-terminal mouse IgG2a (mfNPR1-mFc) (SEQ ID NO: 198), mouse NPR1extracellular domain expressed with a C-terminal mouse IgG2a (mNPR1-mFc)(SEQ ID NO: 199), hNPR1-mFc+hANP, hNPR1-mFc+hBNP, mfNPR1-mFc+hANP,mfNPR1-mFc+hBNP, mNPR1-mFc+mANP, mNPR1-mFc+mBNP. Differentconcentrations of hNPR1-MMH, mfNPR1-MMH (100 nM-3.7 nM, 3-fold serialdilution or 100 nM-6.25 nM, 4-fold serial dilution); hNPR1-mFc,mfNPR1.mFc (100 nM-1.56 nM, 4-fold serial dilution or 100 nM-3.7 nM,3-fold serial dilution); mNPR1.mmh (100 nM), hNPR1-mFc or mfNPR1-mFccomplexed with 10-fold concentration of hANP or hBNP (100 nM, 25 nM,6.25 nM or 100 nM-3.7 nM, 3-fold serial dilution); mNPR1.mFc complexedwith 10-fold concentration of mANP or mBNP (100 nM, 25 nM or 100 nM-3.7nM, 3-fold serial dilution) or hNPR1-hFc or hNPR1-hFc complexed with10-fold concentration of hANP (100 nM-6.25 nM, 4-fold serial dilution)prepared in HBS-ET running buffer, were injected for 4 minutes at a flowrate of 30 μL/min while the dissociation of mAb bound different NPR1reagents was monitored for 10 minutes in HBS-ET running buffer. At theend of each cycle, the NPR1 mAb capture surface was regenerated usingeither 10 sec injection of 20 mM phosphoric acid for mouse anti-human Fcspecific mAb surface or 40 sec injection of 10 mM Glycine, HCl, pH1.5for goat anti-human Fcγ specific polyclonal antibody or two 1-minuteinjections of 10 mM Gly pH1.5. The association rate (k_(a)) anddissociation rate (k_(d)) were determined by fitting the real-timebinding sensorgrams to a 1:1 binding model with mass transportlimitation using Scrubber 2.0c curve-fitting software. Bindingdissociation equilibrium constant (K_(D)) and dissociative half-life(t½) were calculated from the kinetic rates as:

${{K_{D}(M)} = \frac{kd}{ka}},{{{and}\mspace{14mu} t\; \frac{1}{2}\left( \min \right)} = \frac{\ln (2)}{60*{kd}}}$

Results

Binding kinetics parameters for different NPR1 proteins binding toselected anti-NPR1 antibodies of the invention at 25° C. and 37° C. areshown in Tables 3 through 26.

TABLE 3 Binding kinetics parameters of hNPR1-MMH binding to NPR1monoclonal antibodies at 25° C. mAb 100 nM Ag mAb Capture Bound k_(a)k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min) mAb22033 377± 0.9 30 8.55E+03 4.06E−04 4.75E−08 28.4 mAb22035 368 ± 0.6 210 2.03E+051.50E−04 7.38E−10 77.3 mAb22805 356 ± 0.7 0 NB^($) NB^($) NB^($) NB^($)mAb22809 359 ± 1.1 0 NB^($) NB^($) NB^($) NB^($) mAb22810 386 ± 3.8 1NB^($) NB^($) NB^($) NB^($) mAb25479 338 ± 1.2 16 1.40E+04 6.73E−034.82E−07 1.7 mAb25491 275 ± 0.9 37 2.62E+04 9.83E−04 3.76E−08 11.7mAb25497 314 ± 0.6 22 3.17E+04 1.67E−03 5.27E−08 6.9 mAb25498 325 ± 0.868 2.25E+04 1.82E−03 8.10E−08 6.3 mAb25502 349 ± 2.3 2 NB^($) NB^($)NB^($) NB^($) mAb25508 296 ± 1 192 4.32E+05 1.34E−04 3.09E−10 86.5mAb25545 360 ± 0.7 2 NB^($) NB^($) NB^($) NB^($) IgG4 Isotype 315 ± 0.71 NB^($) NB^($) NB^($) NB^($) Control ^($)indicates that no binding wasobserved under the current experimental conditions * indicates that theobserved binding data were inconclusive

TABLE 4 Binding kinetics parameters of hNPR1-MMH binding to NPR1monoclonal antibodies at 37° C. mAb mAb Capture 100 nM Ag k_(a) k_(d) KDt½ Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min) mAb22033 473 ±2.1 52 6.03E+03 8.19E−04 1.36E−07 14.1 mAb22035 492 ± 2.3 284 2.40E+053.12E−04 1.30E−09 37.1 mAb22805 451 ± 2 1 NB^($) NB^($) NB^($) NB^($)mAb22809 466 ± 2 −1 NB^($) NB^($) NB^($) NB^($) mAb22810 428 ± 1.2 1NB^($) NB^($) NB^($) NB^($) mAb25479 443 ± 4.5 16 2.28E+04 1.57E−026.89E−07 0.7 mAb25491 357 ± 1.8 51 3.56E+04 4.53E−03 1.27E−07 2.5mAb25497 402 ± 3 31 1.86E+04 2.58E−03 1.39E−07 4.5 mAb25498 418 ± 2.8 972.25E+04 4.15E−03 1.84E−07 2.8 mAb25502 452 ± 3.3 4 NB^($) NB^($) NB^($)NB^($) mAb25508 397 ± 2.1 252 4.53E+05 2.22E−04 4.90E−10 52.1 mAb25545463 ± 1.7 7 IC* IC* IC* IC* IgG4 Isotype 417 ± 3.1 3 NB^($) NB^($)NB^($) NB^($) Control ^($)indicates that no binding was observed underthe current experimental conditions *indicates that the observed bindingdata were inconclusive

TABLE 5 Binding kinetics parameters of mfNPR1-MMH binding to NPR1monoclonal antibodies at 25° C. mAb 100 nM Ag mAb Capture Bound k_(a)k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min) mAb22033 376± 0.8 79 1.79E+04 2.91E−04 1.62E−08 39.6 mAb22035 368 ± 0.8 245 4.26E+051.43E−04 3.36E−10 80.8 mAb22805 356 ± 1.1 0 NB^($) NB^($) NB^($) NB^($)mAb22809 359 ± 0.9 0 NB^($) NB^($) NB^($) NB^($) mAb22810 381 ± 0.8 1NB^($) NB^($) NB^($) NB^($) mAb25479 338 ± 0.5 41 2.77E+04 6.82E−032.46E−07 1.7 mAb25491 275 ± 0.4 81 4.20E+04 1.28E−03 3.05E−08 9.0mAb25497 315 ± 0.6 49 3.78E+04 2.26E−03 5.98E−08 5.1 mAb25498 327 ± 0.5114 5.79E+04 1.58E−03 2.72E−08 7.3 mAb25502 350 ± 0.9 3 NB^($) NB^($)NB^($) NB^($) mAb25508 298 ± 1.1 213 7.06E+05 1.32E−04 1.87E−10 87.5mAb25545 360 ± 0.5 4 IC* IC* IC* IC* IgG4 Isotype 315 ± 0.6 0 NB^($)NB^($) NB^($) NB^($) Control ^($)indicates that no binding was observedunder the current experimental conditions *indicates that the observedbinding data were inconclusive

TABLE 6 Binding kinetics parameters of mfNPR1-MMH binding to NPR1monoclonal antibodies at 37° C. mAb mAb Capture 100 nM Ag k_(a) k_(d) KDt½ Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min) mAb22033 470 ±1.4 131 2.51E+04 6.97E−04 2.78E−08 16.6 mAb22035 490 ± 1.3 319 5.32E+053.74E−04 7.02E−10 30.9 mAb22805 449 ± 1.3 3 NB^($) NB^($) NB^($) NB^($)mAb22809 465 ± 1 1 NB^($) NB^($) NB^($) NB^($) mAb22810 426 ± 1.1 0NB^($) NB^($) NB^($) NB^($) mAb25479 440 ± 1.8 43 3.96E+04 1.42E−023.60E−07 0.8 mAb25491 353 ± 1.2 106 5.83E+04 4.57E−03 7.84E−08 2.5mAb25497 397 ± 1.4 69 2.77E+04 3.84E−03 1.39E−07 3.0 mAb25498 414 ± 1.7151 7.07E+04 3.69E−03 5.21E−08 3.1 mAb25502 449 ± 1.7 6 NB^($) NB^($)NB^($) NB^($) mAb25508 394 ± 1.1 275 7.82E+05 2.73E−04 3.49E−10 42.3mAb25545 461 ± 1 10 IC* IC* IC* IC* IgG4 Isotype 413 ± 1.3 3 NB^($)NB^($) NB^($) NB^($) Control ^($)indicates that no binding was observedunder the current experimental conditions *indicates that the observedbinding data were inconclusive

TABLE 7 Binding kinetics parameters of mNPR1-MMH binding to NPR1monoclonal antibodies at 25° C. mAb mAb Capture 100 nM Ag k_(a) k_(d) KDt½ Captured Lev el (RU) Bound (RU) (1/Ms) (1/s) (M) (min) mAb22033 376 ±0.7 0 NB^($) NB^($) NB^($) NB^($) mAb22035 368 ± 0.2 0 NB^($) NB^($)NB^($) NB^($) mAb22805 358 ± 1.9 0 NB^($) NB^($) NB^($) NB^($) mAb22809358 ± 0.3 0 NB^($) NB^($) NB^($) NB^($) mAb22810 378 ± 0.3 −1 NB^($)NB^($) NB^($) NB^($) mAb25479 338 ± 1.2 0 NB^($) NB^($) NB^($) NB^($)mAb25491 276 ± 0.3 0 NB^($) NB^($) NB^($) NB^($) mAb25497 315 ± 0.1 0NB^($) NB^($) NB^($) NB^($) mAb25498 326 ± 0.6 0 NB^($) NB^($) NB^($)NB^($) mAb25502 349 ± 0.7 0 NB^($) NB^($) NB^($) NB^($) mAb25508 296 ±1.2 0 NB^($) NB^($) NB^($) NB^($) mAb25545 359 ± 0.1 0 NB^($) NB^($)NB^($) NB^($) IgG4 Isotype 315 ± 0.1 0 NB^($) NB^($) NB^($) NB^($)Control ^($)indicates that no binding was observed under the currentexperimental conditions * indicates that the observed binding data wereinconclusive ^(#)indicates that the conditions were not tested

TABLE 8 Binding kinetics parameters of mNPR1-MMH binding to NPR1monoclonal antibodies at 37° C. mAb 100 nM Ag mAb Capture Bound k_(a)k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min) mAb22033 468± 0.6 1 NB^($) NB^($) NB^($) NB^($) mAb22035 491 ± 0.9 1 NB^($) NB^($)NB^($) NB^($) mAb22805 450 ± 0.2 1 NB^($) NB^($) NB^($) NB^($) mAb22809466 ± 0.1 0 NB^($) NB^($) NB^($) NB^($) mAb22810 420 ± 0.1 −2 NB^($)NB^($) NB^($) NB^($) mAb25479 438 ± 0.2 1 NB^($) NB^($) NB^($) NB^($)mAb25491 353 ± 1.8 0 NB^($) NB^($) NB^($) NB^($) mAb25497 395 ± 0.7 0NB^($) NB^($) NB^($) NB^($) mAb25498 413 ± 0.2 0 NB^($) NB^($) NB^($)NB^($) mAb25502 447 ± 0.9 0 NB^($) NB^($) NB^($) NB^($) mAb25508 392 ±0.3 0 NB^($) NB^($) NB^($) NB^($) mAb25545 460 ± 0.4 1 NB^($) NB^($)NB^($) NB^($) IgG4 Isotype 412 ± 0.4 1 NB^($) NB^($) NB^($) NB^($)Control ^($)indicates that no binding was observed under the currentexperimental conditions * indicates that the observed binding data wereinconclusive ^(#) indicates that the conditions were not tested

TABLE 9 Binding kinetics parameters of hNPR1-mFc or hNPR1-hFc binding toNPR1 monoclonal antibodies at 25° C. mAb mAb Capture 100 nM Ag k_(a)k_(d) KD t½ Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min)mAb22033 351 ± 1.3 56 6.18E+04 6.38E−05 1.03E−09 181.0 mAb22035 347 ±1.2 198 2.25E+05 8.20E−05 3.65E−10 140.9 mAb22805 331 ± 0.8 2 NB^($)NB^($) NB^($) NB^($) mAb22809 327 ± 0.7 −2 NB^($) NB^($) NB^($) NB^($)mAb22810 380 ± 0.4 4 IC* IC* IC* IC* mAb25479 327 ± 2.1 26 3.80E+048.52E−04 2.24E−08 13.6 mAb25491 249 ± 2.8 36 1.01E+05 1.24E−04 1.24E−0992.8 mAb25497 302 ± 25.8 49 2.03E+05 4.83E−05 2.38E−10 239.3 mAb25498312 ± 0.8 94 6.98E+04 2.55E−04 3.65E−09 45.3 mAb25502 321 ± 1.6 178.79E+04 8.74E−05 9.94E−10 132.1 mAb25508 276 ± 1.5 211 7.89E+056.85E−05 8.68E−11 168.6 mAb25545 340 ± 1.1 7 IC* IC* IC* IC* IgG4Isotype 298 ± 1.6 −14 NB^($) NB^($) NB^($) NB^($) Control ^($)indicatesthat no binding was observed under the current experimental conditions*indicates that the observed binding data were inconclusive

TABLE 10 Binding kinetics parameters of hNPR1-mFc or hNPR1-hFc bindingto NPR1 monoclonal antibodies at 37° C. mAb mAb Capture 100 nM Ag k_(a)k_(d) KD t½ Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min)mAb22033 423 ± 3.1 75 3.68E+04 1.31E−04 3.56E−09 88.2 mAb22035 454 ± 3.6257 6.36E+05 1.27E−04 2.00E−10 90.8 mAb22805 406 ± 1.8 8 NB^($) NB^($)NB^($) NB^($) mAb22809 413 ± 1.6 −1 NB^($) NB^($) NB^($) NB^($) mAb22810423 ± 0.8 9 IC* IC* IC* IC* mAb25479 414 ± 5.6 47 3.37E+04 1.39E−034.12E−08 8.3 mAb25491 298 ± 1.6 70 4.19E+04 3.63E−04 8.66E−09 31.8mAb25497 344 ± 1.7 43 4.23E+04 3.10E−04 7.31E−09 37.3 mAb25498 381 ± 2129 8.86E+04 5.68E−04 6.41E−09 20.3 mAb25502 396 ± 2.8 9 NB^($) NB^($)NB^($) NB^($) mAb25508 353 ± 1.3 242 8.38E+05 1.01E−04 1.21E−10 113.9mAb25545 415 ± 2.8 15 IC* IC* IC* IC* IgG4 Isotype 366 ± 3.9 −19 NB^($)NB^($) NB^($) NB^($) Control ^($)indicates that no binding was observedunder the current experimental conditions *indicates that the observedbinding data were inconclusive

TABLE 11 Binding kinetics parameters of hNPR1-mFc:hANP or hNPR1-hFc:hANPcomplex binding to NPR1 monoclonal antibodies at 25° C. mAb 100 nM AgmAb Capture Bound k_(a) k_(d) KD t½ Captured Level (RU) (RU) (1/Ms)(1/s) (M) (min) mAb22033 350 ± 0.4 84 6.03E+04 8.60E−05 1.43E−09 134.4mAb22035 347 ± 0.3 186 1.79E+05 7.78E−05 4.34E−10 148.4 mAb22805 331 ±1.8 84 4.31E+04 1.41E−04 3.28E−09 81.7 mAb22809 327 ± 1.5 53 3.92E+041.93E−04 4.94E−09 59.7 mAb22810 378 ± 0.6 14 8.31E+03 3.44E−04 4.14E−0834 mAb25479 327 ± 2.4 49 3.10E+04 1.09E−04 3.53E−09 105.8 mAb25491 250 ±1.7 71 5.22E+04 4.15E−05 7.94E−10 278.5 mAb25497 292 ± 2.1 59 1.63E+054.56E−05 2.79E−10 253.1 mAb25498 313 ± 0.6 52 4.58E+04 3.17E−04 6.93E−0936.4 mAb25502 321 ± 4.3 81 6.06E+04 2.53E−04 4.17E−09 45.7 mAb25508 278± 4.2 199 2.64E+05 8.97E−05 3.40E−10 128.8 mAb25545 341 ± 1.8 282.34E+04 4.63E−05 1.98E−09 249.7 IgG4 Isotype 297 ± 1.5 -12 NB^($)NB^($) NB^($) NB^($) Control ^($)indicates that no binding was observedunder the current experimental conditions *indicates that the observedbinding data were inconclusive

TABLE 12 Binding kinetics parameters of hNPR1-mFc:hANP or hNPR1-hFc:hANPcomplex binding to NPR1 monoclonal antibodies at 37° C. mAb mAb Capture100 nM Ag k_(a) k_(d) KD t½ Captured Level (RU) Bound (RU) (1/Ms) (1/s)(M) (min) mAb22033 412 ± 1.2 108 5.79E+04 9.80E−05 1.69E−09 117.9mAb22035 444 ± 2 237 2.25E+05 1.14E−04 5.08E−10 101.0 mAb22805 399 ± 1.9113 6.01E+04 2.95E−04 4.91E−09 39.2 mAb22809 406 ± 0.8 68 5.04E+041.66E−04 3.29E−09 69.5 mAb22810 421 ± 0.8 26 1.37E+04 1.04E−03 7.55E−0811 mAb25479 408 ± 7.6 84 4.97E+04 1.34E−04 2.70E−09 86.1 mAb25491 291 ±1.3 96 5.69E+04 1.11E−04 1.95E−09 104.1 mAb25497 336 ± 1.6 60 4.85E+041.02E−04 2.09E−09 113.7 mAb25498 375 ± 1.9 74 5.30E+04 7.20E−04 1.36E−0816.0 mAb25502 388 ± 5.1 105 7.20E+04 4.67E−04 6.49E−09 24.7 mAb25508 348± 0.8 230 2.93E+05 1.48E−04 5.06E−10 77.9 mAb25545 409 ± 2.7 58 3.67E+048.61E−05 2.35E−09 134.2 IgG4 358 ± 4.9 −18 NB^($) NB^($) NB^($) NB^($)Isotype Control ^($)indicates that no binding was observed under thecurrent experimental conditions *indicates that the observed bindingdata were inconclusive

TABLE 13 Binding kinetics parameters of hNPR1-mFc:hBNP complex bindingto NPR1 monoclonal antibodies at 25° C. mAb mAb Capture 100 nM Ag k_(a)k_(d) KD t½ Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min)mAb22033 350 ± 0.4 85 5.43E+04 6.67E−05 1.23E−09 173.2 mAb22035 348 ±2.9 194 1.71E+05 8.70E−05 5.08E−10 132.7 mAb22805 330 ± 0.4 84 4.14E+041.52E−04 3.68E−09 75.8 mAb22809 327 ± 0.4 51 3.28E+04 2.21E−04 6.72E−0952.4 mAb22810 NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 326 ±0.5 50 2.92E+04 1.23E−04 4.21E−09 94.1 mAb25491 250 ± 0.3 72 5.46E+044.96E−05 9.08E−10 232.9 mAb25497 292 ± 0.4 59 1.53E+05 5.49E−05 3.58E−10210.5 mAb25498 313 ± 0.4 55 4.85E+04 2.56E−04 5.28E−09 45.1 mAb25502 317± 1.5 78 6.94E+04 2.36E−04 3.40E−09 49.0 mAb25508 275 ± 0.6 200 2.50E+058.57E−05 3.43E−10 134.7 mAb25545 340 ± 0.2 27 2.30E+04 6.26E−05 2.72E−09184.6 IgG4 Isotype 296 ± 0.5 −11 NB^($) NB^($) NB^($) NB^($) Control^($)indicates that no binding was observed under the currentexperimental conditions * indicates that the observed binding data wereinconclusive ^(#)indicates that the conditions were not tested

TABLE 14 Binding kinetics parameters of hNPR1-mFc:hBNP complex bindingto NPR1 monoclonal antibodies at 37° C. mAb mAb Capture 100 nM Ag k_(a)k_(d) KD t½ Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min)mAb22033 409 ± 1.4 108 5.37E+04 9.60E−05 1.79E−09 120.3 mAb22035 440 ±0.8 243 2.32E+05 1.28E−04 5.53E−10 90.0 mAb22805 398 ± 1.4 112 5.20E+043.13E−04 6.02E−09 36.9 mAb22809 405 ± 0.5 63 4.65E+04 1.66E−04 3.56E−0969.8 mAb22810 NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 402 ±0.4 4.27E+04 1.63E−04 3.83E−09 70.7 mAb25491 289 ± 1.5 96 5.47E+041.34E−04 2.45E−09 86.2 mAb25497 333 ± 1.1 59 4.92E+04 1.16E−04 2.35E−0999.7 mAb25498 372 ± 0.9 76 5.29E+04 9.48E−04 1.79E−08 12.2 mAb25502 391± 0.7 102 6.03E+04 4.72E−04 7.82E−09 24.5 mAb25508 347 ± 0.9 2363.52E+05 1.64E−04 4.66E−10 70.3 mAb25545 405 ± 1.2 56 3.46E+04 9.84E−052.85E−09 117.3 IgG4 Isotype 354 ± 0.4 −17 NB^($) NB^($) NB^($) NB^($)Control ^($)indicates that no binding was observed under the currentexperimental conditions * indicates that the observed binding data wereinconclusive ^(#)indicates that the conditions were not tested

TABLE 15 Binding kinetics parameters of mfNPR1-mFc binding to NPR1monoclonal antibodies at 25° C. mAb 100 nM Ag mAb Capture Bound k_(a)k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min) mAb22033 351± 0.7 96 5.17E+04 4.85E−05 9.38E−10 238.0 mAb22035 346 ± 0.6 2312.44E+05 5.57E−05 2.29E−10 207.2 mAb22805 331 ± 1.6 10 IC* IC* IC* IC*mAb22809 328 ± 0.7 6 NB^($) NB^($) NB^($) NB^($) mAb22810 NT^(#) NT^(#)NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 325 ± 0.7 54 3.12E+04 5.07E−041.62E−08 22.8 mAb25491 251 ± 1.4 81 5.40E+04 7.54E−05 1.40E−09 153.1mAb25497 292 ± 1 76 1.72E+05 5.79E−05 3.37E−10 199.4 mAb25498 313 ± 0.6128 7.20E+04 1.77E−04 2.45E−09 65.4 mAb25502 321 ± 1.9 22 8.83E+041.41E−04 1.60E−09 82.0 mAb25508 276 ± 0.9 230 5.15E+05 5.98E−05 1.16E−10193.3 mAb25545 340 ± 0.7 13 IC* IC* IC* IC* IgG4 Isotype 297 ± 0.6 5NB^($) NB^($) NB^($) NB^($) Control ^($)indicates that no binding wasobserved under the current experimental conditions *indicates that theobserved binding data were inconclusive ^(#)indicates that theconditions were not tested

TABLE 16 Binding kinetics parameters of mfNPR1-mFc binding to NPR1monoclonal antibodies at 37° C. mAb mAb Capture 100 nM Ag k_(a) k_(d) KDt½ Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min) mAb22033 415 ±1.3 126 4.82E+04 9.90E−05 2.06E−09 116.7 mAb22035 447 ± 1.5 291 4.59E+058.44E−05 1.84E−10 136.9 mAb22805 402 ± 1.7 16 IC* IC* IC* IC* mAb22809409 ± 1.6 8 NB^($) NB^($) NB^($) NB^($) mAb22810 NT^(#) NT^(#) NT^(#)NT^(#) NT^(#) NT^(#) mAb25479 408 ± 1.3 89 3.82E+04 1.08E−03 2.81E−0810.7 mAb25491 294 ± 1.6 114 4.70E+04 1.64E−04 3.49E−09 70.4 mAb25497 340± 1.1 79 4.25E+04 2.21E−04 5.20E−09 52.3 mAb25498 377 ± 1.4 170 9.16E+043.72E−04 4.06E−09 31.1 mAb25502 393 ± 1.7 17 IC* IC* IC* IC* mAb25508350 ± 1.1 268 6.34E+05 8.36E−05 1.32E−10 138.2 mAb25545 410 ± 1 265.51E+04 1.44E−04 2.62E−09 80.0 IgG4 360 ± 1.4 8 NB^($) NB^($) NB^($)NB^($) Isotype Control ^($)indicates that no binding was observed underthe current experimental conditions *indicates that the observed bindingdata were inconclusive ^(#)indicates that the conditions were not tested

TABLE 17 Binding kinetics parameters of mfNPR1-mFc:hANP complex bindingto NPR1 monoclonal antibodies at 25° C. mAb 100 nM Ag mAb Capture Boundk_(a) k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min)mAb22033 349 ± 0.5 133 6.58E+04 5.36E−05 8.15E−10 215.5 mAb22035 346 ±0.7 227 1.89E+05 4.66E−05 2.46E−10 247.9 mAb22805 330 ± 0.5 116 4.84E+049.51E−05 1.96E−09 121.4 mAb22809 327 ± 0.5 86 4.47E+04 1.73E−04 3.88E−0966.6 mAb22810 NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 326 ±0.3 86 4.20E+04 1.03E−04 2.44E−09 112.7 mAb25491 250 ± 0.8 111 6.01E+044.55E−05 7.57E−10 253.8 mAb25497 292 ± 0.6 95 1.56E+05 3.75E−05 2.40E−10307.7 mAb25498 313 ± 0.4 81 4.19E+04 1.12E−04 2.68E−09 102.9 mAb25502316 ± 2.7 113 7.40E+04 2.64E−04 3.56E−09 43.8 mAb25508 273 ± 0.5 2243.14E+05 7.32E−05 2.33E−10 157.7 mAb25545 339 ± 0.4 45 2.28E+04 5.37E−052.35E−09 215.1 IgG4 296 ± 0.5 7 NB^($) NB^($) NB^($) NB^($) IsotypeControl ^($)indicates that no binding was observed under the currentexperimental conditions *indicates that the observed binding data wereinconclusive ^(#)indicates that the conditions were not tested

TABLE 18 Binding kinetics parameters of mfNPR1-mFc:hANP complex bindingto NPR1 monoclonal antibodies at 37° C. mAb 100 nM Ag mAb Capture Boundk_(a) k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min)mAb22033 407 ± 1.1 169 6.80E+04 8.90E−05 1.31E−09 129.8 mAb22035 438 ±0.6 283 2.55E+05 7.70E−05 3.02E−10 150.1 mAb22805 395 ± 0.3 153 6.33E+042.09E−04 3.30E−09 55.3 mAb22809 403 ± 0.4 106 5.55E+04 2.05E−04 3.70E−0956.2 mAb22810 NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 401 ±1.6 138 5.47E+04 2.19E−04 4.00E−09 52.8 mAb25491 288 ± 1 148 7.10E+041.06E−04 1.49E−09 109.0 mAb25497 332 ± 0.6 102 5.84E+04 1.52E−042.60E−09 75.9 mAb25498 370 ± 1.5 114 5.01E+04 4.45E−04 8.89E−09 25.9mAb25502 390 ± 0.7 143 9.10E+04 5.24E−04 5.76E−09 22.0 mAb25508 346 ±0.3 262 4.11E+05 1.46E−04 3.55E−10 79.3 mAb25545 404 ± 1.2 86 4.01E+048.18E−05 2.04E−09 141.2 IgG4 Isotype 353 ± 0.8 9 NB^($) NB^($) NB^($)NB^($) Control ^($)indicates that no binding was observed under thecurrent experimental conditions *indicates that the observed bindingdata were inconclusive ^(#)indicates that the conditions were not tested

TABLE 19 Binding kinetics parameters of mfNPR1-mFc:hBNP complex bindingto NPR1 monoclonal antibodies at 25° C. mAb 100 nM Ag mAb Capture Boundk_(a) k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min)mAb22033 349 ± 0.7 132 6.59E+04 5.15E−05 7.81E−10 224.2 mAb22035 346 ±0.3 231 2.05E+05 6.13E−05 3.00E−10 188.3 mAb22805 330 ± 1.5 113 4.53E+049.60E−05 2.12E−09 120.4 mAb22809 327 ± 0.6 80 4.14E+04 1.60E−04 3.85E−0972.4 mAb22810 NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 325 ±0.1 86 3.81E+04 1.13E−04 2.95E−09 102.6 mAb25491 249 ± 0.7 110 5.86E+043.71E−05 6.32E−10 311.7 mAb25497 291 ± 0.1 93 1.40E+05 4.47E−05 3.20E−10258.4 mAb25498 312 ± 0.6 81 4.32E+04 1.09E−04 2.53E−09 106.0 mAb25502314 ± 3.4 110 6.90E+04 2.82E−04 4.08E−09 41.0 mAb25508 273 ± 0.8 2293.08E+05 8.91E−05 2.90E−10 129.6 mAb25545 339 ± 0.1 42 2.39E+04 4.97E−052.08E−09 232.5 IgG4 296 ± 1.1 7 NB^($) NB^($) NB^($) NB^($) IsotypeControl ^($)indicates that no binding was observed under the currentexperimental conditions *indicates that the observed binding data wereinconclusive ^(#)indicates that the conditions were not tested

TABLE 20 Binding kinetics parameters of mfNPR1-mFc:hBNP complex bindingto NPR1 monoclonal antibodies at 37° C. mAb 100 nM Ag mAb Capture Boundk_(a) k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (mm)mAb22033 406 ± 1.4 167 6.75E+04 8.80E−05 1.30E−09 131.3 mAb22035 436 ±1.1 286 2.58E+05 9.48E−05 3.68E−10 121.9 mAb22805 394 ± 1 150 5.96E+042.02E−04 3.39E−09 57.2 mAb22809 402 ± 0.9 99 5.34E+04 2.29E−04 4.29E−0950.5 mAb22810 NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 399 ±0.7 135 5.30E+04 2.93E−04 5.53E−09 39.4 mAb25491 286 ± 0.7 146 6.89E+041.10E−04 1.60E−09 105.0 mAb25497 330 ± 0.5 100 5.74E+04 1.66E−042.90E−09 69.5 mAb25498 369 ± 0 113 5.21E+04 4.64E−04 8.91E−09 24.9mAb25502 388 ± 1.5 139 8.52E+04 4.81E−04 5.65E−09 24.0 mAb25508 344 ±0.3 266 4.23E+05 1.50E−04 3.54E−10 77.1 mAb25545 402 ± 0.7 82 3.71E+047.77E−05 2.09E−09 148.6 IgG4 Isotype 351 ± 0.5 9 NB^($) NB^($) NB^($)NB^($) Control ^($)indicates that no binding was observed under thecurrent experimental conditions *indicates that the observed bindingdata were inconclusive ^(#)indicates that the conditions were not tested

TABLE 21 Binding kinetics parameters of mNPR1-mFc binding to NPR1monoclonal antibodies at 25° C. mAb 100 nM Ag mAb Capture Bound k_(a)k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min) mAb22033 350± 0.5 0 NB^($) NB^($) NB^($) NB^($) mAb22035 346 ± 0.8 −1 NB^($) NB^($)NB^($) NB^($) mAb22805 330 ± 0.5 0 NB^($) NB^($) NB^($) NB^($) mAb22809328 ± 0.7 0 NB^($) NB^($) NB^($) NB^($) mAb22810 NT^(#) NT^(#) NT^(#)NT^(#) NT^(#) NT^(#) mAb25479 325 ± 0.3 −1 NB^($) NB^($) NB^($) NB^($)mAb25491 250 ± 1.1 1 NB^($) NB^($) NB^($) NB^($) mAb25497 292 ± 0.7 1NB^($) NB^($) NB^($) NB^($) mAb25498 313 ± 1 0 NB^($) NB^($) NB^($)NB^($) mAb25502 321 ± 1.1 1 NB^($) NB^($) NB^($) NB^($) mAb25508 275 ±0.7 −1 NB^($) NB^($) NB^($) NB^($) mAb25545 340 ± 0.2 0 NB^($) NB^($)NB^($) NB^($) IgG4 Isotype 296 ± 0.6 0 NB^($) NB^($) NB^($) NB^($)Control ^($)indicates that no binding was observed under the currentexperimental conditions ^(#)indicates that the conditions were nottested ^(#)indicates that the conditions were not tested

TABLE 22 Binding kinetics parameters of mNPR1-mFc binding to NPR1monoclonal antibodies at 37° C. mAb 100 nM Ag mAb Capture Bound k_(a)k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (mm) mAb22033 413± 0.4 1 NB^($) NB^($) NB^($) NB^($) mAb22035 444 ± 0.5 0 NB^($) NB^($)NB^($) NB^($) mAb22805 400 ± 0.2 0 NB^($) NB^($) NB^($) NB^($) mAb22809407 ± 1 0 NB^($) NB^($) NB^($) NB^($) mAb22810 NT^(#) NT^(#) NT^(#)NT^(#) NT^(#) NT^(#) mAb25479 406 ± 0.3 2 NB^($) NB^($) NB^($) NB^($)mAb25491 291 ± 0.9 1 NB^($) NB^($) NB^($) NB^($) mAb25497 336 ± 0.7 1NB^($) NB^($) NB^($) NB^($) mAb25498 375 ± 1 0 NB^($) NB^($) NB^($)NB^($) mAb25502 391 ± 0.6 1 NB^($) NB^($) NB^($) NB^($) mAb25508 347 ±0.4 0 NB^($) NB^($) NB^($) NB^($) mAb25545 407 ± 0.7 1 NB^($) NB^($)NB^($) NB^($) IgG4 Isotype 359 ± 0.6 2 NB^($) NB^($) NB^($) NB^($)Control ^($)indicates that no binding was observed under the currentexperimental conditions ^(#)indicates that the conditions were nottested

TABLE 23 Binding kinetics parameters of mNPR1-mFc:hANP complex bindingto NPR1 monoclonal antibodies at 25° C. mAb 100 nM Ag mAb Capture Boundk_(a) k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min)mAb22033 348 ± 0.4 1 NB^($) NB^($) NB^($) NB^($) mAb22035 345 ± 0.4 0NB^($) NB^($) NB^($) NB^($) mAb22805 330 ± 1.1 18 1.98E+05 8.39E−024.24E−07 0.14 mAb22809 326 ± 0.4 0 NB^($) NB^($) NB^($) NB^($) mAb22810NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 324 ± 0.8 1 NB^($)NB^($) NB^($) NB^($) mAb25491 249 ± 0.9 1 NB^($) NB^($) NB^($) NB^($)mAb25497 290 ± 0.5 1 NB^($) NB^($) NB^($) NB^($) mAb25498 311 ± 0.9 0NB^($) NB^($) NB^($) NB^($) mAb25502 317 ± 2.4 25 2.37E+05 7.99E−023.37E−07 0.14 mAb25508 274 ± 0.8 0 NB^($) NB^($) NB^($) NB^($) mAb25545339 ± 0.3 1 NB^($) NB^($) NB^($) NB^($) IgG4 Isotype 295 ± 0.8 1 NB^($)NB^($) NB^($) NB^($) Control ^($)indicates that no binding was observedunder the current experimental conditions ^(#)indicates that theconditions were not tested

TABLE 24 Binding kinetics parameters of mNPR1-mFc:hANP complex bindingto NPR1 monoclonal antibodies at 37° C. mAb 100 nM Ag mAb Capture Boundk_(a) k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min)mAb22033 402 ± 1.8 2 NB^($) NB^($) NB^($) NB^($) mAb22035 435 ± 0.8 1NB^($) NB^($) NB^($) NB^($) mAb22805 392 ± 1 15 IC* IC* IC* IC* mAb22809400 ± 0.7 1 NB^($) NB^($) NB^($) NB^($) mAb22810 NT^(#) NT^(#) NT^(#)NT^(#) NT^(#) NT^(#) mAb25479 397 ± 1.2 2 NB^($) NB^($) NB^($) NB^($)mAb25491 284 ± 0.7 1 NB^($) NB^($) NB^($) NB^($) mAb25497 328 ± 0.8 1NB^($) NB^($) NB^($) NB^($) mAb25498 367 ± 0.4 1 NB^($) NB^($) NB^($)NB^($) mAb25502 386 ± 2.1 35 2.64E+05 8.83E−02 3.34E−07 0.13 mAb25508343 ± 1.1 0 NB^($) NB^($) NB^($) NB^($) mAb25545 401 ± 0.8 3 NB^($)NB^($) NB^($) NB^($) IgG4 Isotype 349 ± 0.5 2 NB^($) NB^($) NB^($)NB^($) Control ^($)indicates that no binding was observed under thecurrent experimental conditions *indicates that the observed bindingdata were inconclusive ^(#)indicates that the conditions were not tested

TABLE 25 Binding kinetics parameters of mNPR1-mFc:hBNP complex bindingto NPR1 monoclonal antibodies at 25° C. mAb mAb Capture 100 nM Ag k_(a)k_(d) KD t½ Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min)mAb22033 347 ± 1.1 5 NB^($) NB^($) NB^($) NB^($) mAb22035 345 ± 0.8 0NB^($) NB^($) NB^($) NB^($) mAb22805 329 ± 0.9 21 1.53E+05 6.82E−024.45E−07 0.17 mAb22809 326 ± 1.1 1 NB^($) NB^($) NB^($) NB^($) mAb22810NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 325 ± 0.2 1 NB^($)NB^($) NB^($) NB^($) mAb25491 249 ± 0.4 5 NB^($) NB^($) NB^($) NB^($)mAb25497 291 ± 0.2 2 NB^($) NB^($) NB^($) NB^($) mAb25498 312 ± 0.8 1NB^($) NB^($) NB^($) NB^($) mAb25502 318 ± 0.3 25 2.34E+05 5.03E−022.15E−07 0.23 mAb25508 274 ± 0.6 1 NB^($) NB^($) NB^($) NB^($) mAb25545339 ± 0.4 3 NB^($) NB^($) NB^($) NB^($) IgG4 Isotype 296 ± 0.6 2 NB^($)NB^($) NB^($) NB^($) Control ^($)indicates that no binding was observedunder the current experimental conditions. ^(#)indicates that theconditions were not tested

TABLE 26 Binding kinetics parameters of mNPR1-mFc:hBNP complex bindingto NPR1 monoclonal antibodies at 37° C. mAb 100 nM Ag mAb Capture Boundk_(a) k_(d) KD t½ Captured Level (RU) (RU) (1/Ms) (1/s) (M) (min)mAb22033 401 ± 0.2 4 NB^($) NB^($) NB^($) NB^($) mAb22035 433 ± 0.5 1NB^($) NB^($) NB^($) NB^($) mAb22805 389 ± 0.8 17 IC* IC* IC* IC*mAb22809 397 ± 1.3 1 NB^($) NB^($) NB^($) NB^($) mAb22810 NT^(#) NT^(#)NT^(#) NT^(#) NT^(#) NT^(#) mAb25479 397 ± 0.9 2 NB^($) NB^($) NB^($)NB^($) mAb25491 282 ± 0.9 4 NB^($) NB^($) NB^($) NB^($) mAb25497 327 ±0.5 2 NB^($) NB^($) NB^($) NB^($) mAb25498 366 ± 0.6 1 NB^($) NB^($)NB^($) NB^($) mAb25502 384 ± 1.3 34 2.91E+05 9.98E−02 3.42E−07 0.12mAb25508 342 ± 0.9 1 NB^($) NB^($) NB^($) NB^($) mAb25545 400 ± 0.4 4NB^($) NB^($) NB^($) NB^($) IgG4 Isotype 348 ± 0.7 3 NB^($) NB^($)NB^($) NB^($) Control ^($)indicates that no binding was observed underthe current experimental conditions *indicates that the observed bindingdata were inconclusive ^(#)indicates that the conditions were not tested

As shown in Tables 3 to 6, selected anti-NPR1 antibodies bound tomonomeric human and monkey NPR1.

As shown in Tables 9 to 20, the antibodies bound to human and monkeyNPR1 dimer in both the presence and absence of ANP or BNP.

The antibodies did not bind to mouse NPR1, except mAb25502 bound tomNPR1 in the presence of ANP/BNP (Tables 21 to 26).

Example 4: Cross-Competition Between Selected Anti-NPR1 AgonistMonoclonal Antibodies Experimental Procedure

Binding competition within a panel of anti-NPR1 monoclonal antibodieswas determined using a real time, label-free bio-layer interferometryassay on the Octet HTX biosensor platform (Pall ForteBio Corp.). Theentire experiment was performed at 25° C. in 10 mM HEPES, 150 mM NaCl, 3mM EDTA, and 0.05% v/v Surfactant Tween-20, 1 mg/mL BSA, pH7.4 (HBS-EBT)buffer with the plate shaking at the speed of 1000 rpm. To assesswhether 2 antibodies are able to compete with one another for binding totheir respective epitopes, 100 nM recombinant human NPR1 expressed witha C-terminal mouse IgG2a (hNPR1-mFc; SEQ ID: 453) was first incubatedwith 2 μM of human ANP for at least 2 hours. Around 0.3-0.5 nmrecombinant hNPR1-mFc/hANP complex was first captured by dippinganti-mouse Fc antibody coated Octet biosensor tips (Fortebio Inc,#18-5090) by submerging the biosensor tips for 45 seconds in wellscontaining hNPR1-mFc/hANP complex. The antigen captured biosensor tipswere then saturated with the first anti-NPR1 monoclonal antibody(referred to as mAb-1) by dipping into wells containing 50 μg/mLsolution of mAb-1 for 5 minutes. The biosensor tips were then dippedinto wells containing 50 μg/mL solution of second anti-NPR1 monoclonalantibody (referred to as mAb-2) for 3 minutes. The biosensor tips werewashed in HBS-EBT buffer between every step of the experiment. Thereal-time binding response was monitored during the entire course of theexperiment and the binding response at the end of every step wasrecorded. The response of mAb-2 binding to hNPR1-mFc/hANP pre-complexedwith mAb-1 was compared and competitive/non-competitive behavior ofdifferent anti-NPR1 monoclonal antibodies was determined.

Results

TABLE 27 Cross-competition between anti-NPR1 monoclonal antibodies mAb-2competing with mAb-1 mAb-1 mAb25498 mAb25508 mAb25508 mAb25498 mAb22809mAb22810 mAb22033 mAb25491 mAb25497 mAb25502 mAb25545 mAb22805 mAb25479mAb22033 mAb22809 mAb25491 mAb25497 mAb25502 mAb25545 mAb22805 mAb25479mAb22810 mAb25491 mAb22809 mAb22033 mAb25497 mAb25502 mAb25545 mAb22805mAb25479 mAb22810 mAb25497 mAb22809 mAb22033 mAb25491 mAb25502 mAb25545mAb22805 mAb25479 mAb22810 mAb25502 mAb22809 mAb22033 mAb25491 mAb25497mAb25545 mAb22805 mAb25479 mAb22810 mAb25545 mAb22809 mAb22033 mAb25491mAb25497 mAb25502 mAb22805 mAb25479 mAb22810 mAb22805 mAb22809 mAb22033mAb25491 mAb25497 mAb25502 mAb25545 mAb22810 mAb25479 mAb22809 mAb22033mAb25491 mAb25497 mAb25502 mAb25545 mAb22810 mAb22810 mAb22809 mAb25445

Table 27 shows the cross-competition between selected anti-NPR1antibodies.

Example 5: Antibody Binding to Cells Expressing NPR1 ExperimentalProcedure

The ability of anti-human (h) NPR1 monoclonal antibodies to bind tohuman NPR1 (hNPR1) expressing cells with or without one of theligands—human ANP (hANP), was determined using electrochemiluminescence(ECL) based detection.

Briefly, HEK293/hNPR1.myc.DKK expressing cells were engineered bytransfecting human embryonic kidney (HEK) 293 cells with neomycinresistant pLVX.hNPR1.myc.DDK expression plasmid encoding human NPR1(amino acids M1-G1061, UniProtKB-P16066). The non-transfected HEK293cells have no detectable expression of NPR1 by fluorescence activatedcell sorting (FACS) with commercial a-hNRP1 antibodies and were includedas non-specific binding controls.

Experiments were carried out according to the following procedure. Cellsfrom lines described above were rinsed once in 1×PBS buffer withoutCa²⁺/Mg²⁺ and incubated for 10 minutes at 37° C. with Enzyme Free CellDissociation Solution to detach cells from a flask. All cells werewashed once with 1×PBS with Ca²⁺/Mg²⁺ and counted with a Cellometer™Auto T4 cell counter (Nexcelom Bioscience). Approximately 2.0×10⁴HEK293/hNPR1.myc.DDK or HEK293 cells were seeded separately onto 96-wellcarbon electrode plates (MULTI-ARRAY high bind plate, Meso ScaleDiscovery (MSD, Rockville, Md.)) and incubated for 1 hour (h) at 37° C.Nonspecific binding sites were blocked by 2% BSA (w/v) in 1×PBS withCa²⁺/Mg²⁺ for 1 h at room temperature (RT). HEK293/hNPR1.myc.DDK cellswere incubated for 0.5 hour at RT in sample dilution buffer with orwithout 10 nM human ANP (Tocris, Minneapolis, Minn.), and HEK293 cellswere incubated in sample dilution buffer only at the same conditions.Without washing, serial dilutions of anti-NPR1, COMP1 or isotype controlantibodies, ranging from 1.7 μM to 100 nM, or buffer containing noantibody were added to plate-bound cells for 1 h, RT. Plates were thenwashed to remove unbound antibodies and/or hANP using an AquaMax2000plate washer with a cell washing head (MDS Analytical Technologies,Sunnyvale, Calif.). The plate-bound antibodies were detected withSULFO-TAG™-conjugated goat polyclonal anti-human IgG antibody specificfor heavy and light chains (Jackson Immunoresearch, West Grove, Pa.) for1 h, RT.

After washes, plates were developed with Read Buffer (MSD, Rockville,Md.) according to manufacturer's recommended procedure and luminescentsignals were recorded with a SECTOR Imager 600 (MSD, Rockville, Md.).Luminescence intensity, measured in relative light units (RLU), wasrecorded to indicate the binding intensity of each antibody at the rangeof concentrations. The ratio of signal detected with 3.7 nM antibodybinding to the NPR1 engineered cells with or without 10 nM hANP comparedto the same concentration of antibody binding to parental cells with nohANP added was reported as an indication of specificity of NPR1 binding.Antibodies with the binding ratio of greater than or equal to 3 wereclassified as specific binders and antibodies with the binding ratioless than 3 were classified as non-binders.

In addition, direct binding signals (RLU) were analyzed as a function ofthe antibody concentration and data were fitted with a sigmoidal(four-parameter logistic) dose-response model using R statisticalpackage (open source). The EC₅₀ value, defined as the concentration ofantibody at which 50% of the maximal binding signal is detected, wasdetermined to indicate binding potency to NPR1 engineered cells with orwithout 10 nM hANP. EC₅₀ values were reported only for specific bindersand marked with (-) for antibodies with ratio below 3 in Table 28.

Results

Table 28 shows binding of selected anti-NPR1 antibodies to cellsengineered to express human NPR1 or NPR1-ANP complex.

TABLE 28 Anti-NPR1 Antibodies Binding to Cells Engineered to ExpressHuman NPR1 or NPR1-ANP Complex on the Cell Surface Ratio at 3.7 nM Abconcentration of Cell Binding Signal (RLU) to HEK293/ hNPR1.myc.DKK CellBinding Potency to relative to HEK293/hNPR1.myc.DKK, parental HEK293EC₅₀ (M) Ab PID +10 nM hANP 0 hANP +10 nM hANP 0 hANP Peptide-dependentNPR1 binders mAb22809 166 2 4.8E−10 — mAb22805 222 1 6.5E−10 — mAb2554579 1 1.9E−09 — mAb22810 24 1 3.7E−09 — mAb25502 14 1 INC — Comparator 1103 2 1.5E−10 — Peptide-independent NPR1 binders mAb22033 236 2078.2E−10 4.9E−10 mAb25497 87 50 8.7E−10 1.5E−09 mAb22035 220 151 8.9E−102.3E−09 mAb25491 78 58 1.0E−09 1.6E−09 mAb25479 51 30 1.2E−09 1.7E−09mAb25508 35 18 1.3E−09 INC mAb25498 49 41 1.8E−09 4.6E−09 hIgG4 Isotype1 1 — — Control (INC) = inconclusive, no top plateau developed withfour-parameters sigmoidal curve fit to calculate EC₅₀ value, butantibody specifically bound to NPR1 engineered cells with or without 10nM hANP with ratio of equal or greater than 3

As the results in Table 28 show, all anti-NPR1 antibodies boundspecifically to the hNPR1 engineered cells in the presence of 10 nM hANPwith a ratio ≥2. The potency of these antibodies on HEK293/hNPR1.myc.DDKcells with 10 nM hANP ranged from EC₅₀ values of 0.15 nM to 3.7 nM. Fiveantibodies and Comparator) specifically bound to NPR1 engineered cellsonly in the presence of 10 nM hANP and were classified aspeptide-dependent NPR1 binders. The other seven antibodies bound tohNPR1 engineered cells with and without 10 nM hANP and these antibodieswere classified as peptide-independent NPR1 binders. The potency ofthese antibodies on HEK293/hNPR1.myc.DDK cells with no hANP added rangedfrom EC₅₀ values of 0.49 nM to 4.6 nM.

Example 6: Activation of NPR1 by Agonist Anti-NPR1 Monoclonal AntibodiesExperimental Procedure

In order to assess the transcriptional activation of human natriureticpeptide receptor 1 (hNPR1), a stable cell line was developed by stablyexpressing cyclic nucleotide gated channel alpha 2 (CNGA2) and hNPR1 inHEK293. CNGA2 is a calcium channel that can be activated by cGMP,therefore, it can be used as a sensor for cGMP generation (Wunder et al.2005, PMID: 15766716). As NPR1 is activated by the ligand and producescGMP (Zois et al., 2014, PMID: 24820868), CNGA2 is activated and calciuminflux can be measured using a fluorescent Ca⁺⁺ indicator. The cell linewas sorted for high expression of hNPR1, HEK293/hNPR1.MycDDK/CNGA2.MycHS or abbreviated as HEK293/CNGA2/hNPR1, and maintained in DMEMcontaining 10% FBS, NEAA, pen/strep/glut, 100 μg/mL hygromycin, and 500μg/mL G418 sulfate.

A bioassay was performed to measure the effect of anti-hNPR1 antibodieson human NPR1 signaling in the absence of ANP. For the bioassay,HEK293/CNGA2/hNPR1 cells were seeded in black clear bottom PDL plates at30,000 cells/well in DMEM containing 10% FBS, NEAA, pen/strep/glut andincubated at 37° C. and 5% CO₂ overnight. The next morning, media wasremoved and cells were loaded with 80 μl of FLUO4-NW assay buffer withprobenecid (Thermo Scientific) for 30 min at 37° C. Purified hNPR1antibodies or an isotype control antibody were serially diluted at 1:3from ˜0.4 nM-1 μM (8-point) or ANP at 1:3 from ˜0.3 μM to 2 nM(10-point) were transferred to the assay plate using FLIPR TETRA®(Molecular Devices). Base line and response images were captured. Dosageresponse curve were determined based on Max-Min or Area under curve(shown here) and the results were analyzed using nonlinear regression(4-parameter logistics) with Prism™6 software (GraphPad) to obtain EC₅₀values. The % activation of antibodies was calculated with the maximumrange of RFU achieved by the antibody over the maximum range of RFUachieved by ANP.

Results

Table 29 shows activation of human NPR1 by anti-NPR1 antibodies.

TABLE 29 Activation of human NPR1 by anti-NPR1 antibodies Expt 1 Expt IICells 293/CNGA2/hNPR1 293/CNGA2/hNPR1 Mode Activation Activation LigandANP ANP EC₅₀ (pM) 60.5-98.8 78.8 Constant None None % Activation %Activation (vs. ANP Antibody EC₅₀ (nM) (vs ANP max) EC₅₀ (nM) max)mAb22033 83.7 129.54 41.6 129.20% mAb22810 Not tested Not tested 50.8125.50% mAb25545 187.9 123.14% 74.1 122.70% mAb25502 161.4 92.90% >200109.80% mAb22805 239 91.61% 112.3 118.90% mAb22809 270 86.72% Not testedNot tested mAb25491 101.8 74.64% 43.4 103.70% mAb22035 383.9 73.07% >20056.80% mAb25497 72.62 71.09% 56.4 103.40% mAb25498 335.3 67.74% Nottested Not tested mAb25479 174.3 66.73% 101.9 82.80% mAb25508 325.756.08% Not tested Not tested Comparator 1 >2000 53.45% No activation13.80% Isotype No −6.03% No activation 1.90% Control activation

The activation of hNPR1 by selected anti-NPR1 antibodies of theinvention was tested by measuring calcium flux activity inHEK293/CNGA2/hNPR1 in two experiments (Expt I and II)(Table 29). Asshown in Table 29 (in Expt I), 11 purified NPR1 antibodies showedactivation in Ca2+ flux, ranging from 55-130% maximum activation withEC₅₀s ranging 83.7-383.9 nM. 19 antibodies showed weak activation withmaximum activation less than 31% (data not shown). In Expt II, 9anti-NPR1 antibodies showed activation in Ca2+flux ranging from 57-129%maximum activation with EC₅₀s of 41.6->200 nM (Table 29). ANP activatedwith EC₅₀s of 60.5-98.8 pM. No activation was observed with the controlhIgG4 isotype antibody.

Example 7: Effect of a Single Dose of an Agonist Anti-NPR1 MonoclonalAntibody on Systemic Blood Pressure in Normotensive NPR1^(hu/hu) MiceExperimental Procedure

The objectives of this study were to assess the effects of selected NPR1agonist antibodies on baseline systemic blood pressure in telemeterednormotensive NPR1^(hu/hu) mice. Male NPR1^(hu/hu) (n=50) mice aged˜10-20 weeks were implanted with PA-C10 telemeters (DSI, St. Paul,Minn.) and allowed to recover for 7 days, prior to being assigned togroup (Groups 1-12) (Table 30).

TABLE 30 Summary of doses and dose groups Dose Group Test or ControlDose Level Volume Number of Animals No. Article (mg/kg/dose) (mL/kg)Males 1 PBS control 25 5 9 3 mAb22035 5 4 mAb22033 4 5 mAb25497 3 6mAb22805 4 7 mAb25545 3 8 mAb22809 5 9 mAb25479 4 10 mAb25491 4 11mAb25502 5 12 mAb22810 4

Animals were individually housed under standard conditions (Temperaturesof 64° F. to 84° F. (18° C. to 29° C.); relative humidity of 30% to 70%)and a 12-hour light/12-hour dark cycle was maintained. Food (ResearchDiets Standard pellet chow) and water were provided ad libitum.

The test proteins or phosphate buffered saline (PBS) were administeredto the appropriate animals by single subcutaneous injection on Day 0.The dose volume for each animal was based on the most recent body weightmeasurement.

Systolic pressure, diastolic pressure, mean arterial pressure and heartrate were collected for 10 seconds every minute for the duration of thetesting period. An acute assessment of efficacy was made with datacompiled from study day 3 to study day 7. The chronic effects of NPR1agonist antibodies were assessed with daily 24-hour data collected andaveraged over 28 days. All data are presented as mean±SEM.

Results

The initial in vivo screen of NPR1 agonist antibodies demonstrated thatwhen compared to PBS-dosed control animals from days 3 to 7 post-dose, 9antibodies (mAb22033, mAb25479, mAb25497, mAb22805, mAb25545, mAb22809,mAb25491, mAb25502 and mAb22810) significantly reduced, and 1 antibody(mAb22035) had no effect on systemic blood pressures (FIG. 1). Themagnitude of blood pressure reduction as assessed by mean (of days 3 to7 post-dose) systolic blood pressure change from baseline ranged from−3.2±0.2 (mAb25497N) to −11.5±0.8 (mAb22810) mmHg.

Chronic effects of the NPR1 agonist antibodies were evaluated over 28days (Table 31).

TABLE 31 28-day mean blood pressures and heart rates Mean Arterial HeartRate Dose Group Systolic (mmHg) Diastolic (mmHg) (mmHg) (BPM) PBScontrol 0.08 ± 0.09 1.09 ± 0.25 1.19 ± 0.27 11.79 ± 3.17  mAb25545 −4.34± 0.18*   0.13 ± 0.38*  −2.0 ± 0.46*  5.35 ± 3.13* mAb22805 −3.90 ±0.17* −1.51 ± 0.30* −3.40 ± 0.40* 18.83 ± 2.73* mAb25497 −3.04 ± 0.20*  2.37 ± 0.36* 1.04 ± 0.44 12.07 ± 2.64* mAb22035   3.80 ± 0.32*   2.05± 0.22*  2.95 ± 0.26*  2.86 ± 2.33* mAb22033 −11.07 ± 0.59*  −4.55 ±0.38* −8.01 ± 0.48* −2.14 ± 2.83* mAb22810 −7.88 ± 0.57* −2.24 ± 0.31*−5.21 ± 0.42* −6.10 ± 2.96* mAb25502 −3.63 ± 0.49* −0.41 ± 0.40* −2.13 ±0.44* −14.27 ± 2.87*  mAb22809 −2.01 ± 0.58*   2.95 ± 0.70* 0.30 ± 0.62 3.95 ± 2.33* mAb25491 −4.90 ± 0.35* −1.22 ± 0.26* −3.19 ± 0.30* −12.9 ±3.22* mAb25479 −3.44 ± 0.50*   0.51 ± 0.42* −1.60 ± 0.44* −4.01 ± 3.03*Telemetered normotensive NPR1^(hu/hu) mice were randomized into groupsbased off of body weight. Animals were given a single 25 mg/kgsubcutaneous injection of NPR1 agonist mAb or PBS control as describedin Table 1. All values are mean ± SEM, n = 3-9 per group. Statistics -two way ANOVA with Dunnett's; *p < .05 vs. PBS control

Following a single subcutaneous injection on day 0, one antibody(mAb22035) significantly increased pressures by ˜4-7 mmHg, while theremaining antibodies reduced systemic blood pressures 2 to 11 mmHg innormotensive NPR1^(hu/hu) mice. Heart rate responses to both increasedand decreased systemic blood pressures were variable, with some groupsincreasing and others decreasing.

Example 8: Dose Effect of an Agonist Anti-NPR1 Monoclonal Antibody onSystemic Blood Pressure in Normotensive NPR1^(hu/hu) Mice ExperimentalProcedure

The objective of this study was to assess a dose response of the NPR1agonist antibody mAb22033 on systemic blood pressure in telemeterednormotensive NPR1^(hu/hu) mice. Male NPR1^(hu/hu) (n=30) mice aged ˜20weeks were implanted with PA-C10 telemeters (DSI, St. Paul, Minn.) andallowed to recover for 7 days, prior to being assigned to group (Groups1-5) (Table 32).

TABLE 32 Summary of doses and dose groups Dose Number Group Dose LevelVolume of Animals No. Test or Control Article (mg/kg/dose) (mL/kg) Males1 IgG4 isotype control 25 5 6 2 mAb22033 1 5 7 3 mAb22033 5 5 6 4mAb22033 25 5 7 5 mAb22033 50 5 7

Animals were individually housed under standard conditions (Temperaturesof 64° F. to 84° F. (18° C. to 29° C.); relative humidity of 30% to 70%)and a 12-hour light/12-hour dark cycle was maintained. Food (ResearchDiets Standard pellet chow) and water were provided ad libitum.

The test proteins were administered to the appropriate animals once onday 0 via subcutaneous injection. The dose volume for each animal wasbased on the most recent body weight measurement. For urine and serumbiomarker assessment, urine was collected on day 28 and blood sampleswere collected on study day 14 and upon termination. Echocardiographywas performed on day 28, prior to diuresis.

Systolic pressure, diastolic pressure, mean arterial pressure, and heartrate were collected for 10 seconds every minute for the duration of thetesting period. Graphically displayed telemetry data were obtained fromanimals with viable signals for the duration of the in-life portion ofthe study.

Results

Blood pressures (FIGS. 2, 3 and 5) were reduced 10-15 mmHg for up to 4weeks after a single dose of mAb22033 to normotensive NPR1^(hu/hu) mice.Peak pressure reductions for all doses were similar, with the durationof blood pressure effect being approximately 10 days at 1 mg/kg andlonger than 28 days for 50 mg/kg.

TABLE 33 28-day Mean Blood Pressures and Heart Rate Systolic DiastolicMean Arterial Heart Rate Dose Group: (mmHg) (mmHg) (mmHg) (BPM) IgG4isotype control (25   126 ± 0.3 97.0 ± 0.3 112.2 ± 0.3 524.9 ± 3.2mg/kg) mAb22033 (1 mg/kg) 124.4 ± 0.5 95.1 ± 0.4 110.4 ± 0.4 530.6 ± 4.0mAb22033 (5 mg/kg) 120.0 ± 0.4**** 93.8 ± 0.3** 107.6 ± 0.3**** 543.4 ±4.0**** mAb22033 (25 mg/kg) 119.7 ± 0.5**** 93.7 ± 0.4* 107.1 ± 0.4****524.5 ± 3.4 mAb22033 (50 mg/kg) 118.5 ± 0.5**** 94.2 ± 0.3* 106.8 ±0.3**** 553.6 ± 3.5**** Telemetered normotensive NPR1^(hu/hu) mice wererandomized into five groups of equal body weight and given a singlesubcutaneous injection of mAb22033 at the doses listed in Table 1. AnIgG4 antibody was used as isotype control. All values are mean ± SEM, n= 6-7 per group. Statistics - two way ANOVA with Dunnett's; *p < .05 vs.IgG4 isotype control, **p < .01 vs. IgG4 isotype control; ***p < .001vs. IgG4 isotype control; ****P < .0001 vs. IgG4 isotype control

Urinary cGMP concentrations (Table 34) were significantly increased onday 28 in the 50 mg/kg group, with all other doses being of a similarlevel to controls.

TABLE 34 Urine and serum markers Urinary Serum Urine Volume cGMPNTproANP Serum NTproANP Dose Group: (mL/day) (mmol/day) (nmol/L) - Day14 (nmol/L) - Day 29 IgG4 isotype control (25 1.4 ± 0.2 5.0 ± 1.3 0.21 ±.06  0.49 ± 0.17 mg/kg) mAb22033 (1 mg/kg) 1.2 ± 0.2 5.2 ± 1.3 0.35 ±0.11 0.52 ± 0.12 mAb22033 (5 mg/kg) 0.9 ± 0.1 3.7 ± 0.8 0.20 ± 0.08 0.20± 0.05 mAb22033 (25 mg/kg) 1.2 ± 0.2 5.9 ± 0.8 0.14 ± 0.06 0.24 ± 0.06mAb22033 (50 mg/kg) 1.4 ± 0.2  11.9 ± 2.1** 0.26 ± 0.08 0.56 ± 0.20Telemetered normotensive NPR1^(hu/hu) mice were randomized into fivegroups of equal body weight and given a single subcutaneous injection ofmAb22033 at the doses listed in Table 1. An IgG4 antibody was used asisotype control. All values are mean ± SEM, n = 6-7 per group.Statistics - one way ANOVA with Dunnett's; **p < .01 vs. IgG4 isotypecontrol

Relative heart weight (Table 35) was lower in the 50 mg/kg group, withtrending reductions in the 1, 5 and 25 mg/kg groups. No significanteffects on body weight (Table 35), absolute heart weight (Table 35),standard serum (ALT, AMYLAS, AST, CHOL, CKNAC, CREA, DHDL, IP, TRIG, TP,UA, UN, MG, NEFA) or urine (ALB, GLUH, CREA, PRO, CA, IP, MG, UN,AMYLAS, UA, NA, K, CL) chemistries were observed.

TABLE 35 Terminal Body and Organ Weights Terminal Heart Heart Body HeartWeight:Tibia Weight:Body Dose Group: Weight (g) Weight (mg) LengthWeight IgG4 isotype 33.1 ± 1.7 0.143 ± 0.003 0.0079 ± 0.0002 0.0043 ±0.0001 control (25 mg/kg) mAb22033 (1 mg/kg) 34.5 ± 2.3 0.140 ± 0.0050.0078 ± 0.0002 0.0041 ± 0.0001 mAb22033 (5 mg/kg) 34.3 ± 1.4 0.131 ±0.006 0.0075 ± 0.0004 0.0039 ± 0.0003 mAb22033 (25 mg/kg) 32.4 ± 1.40.126 ± 0.006 0.0071 ± 0.0003 0.0039 ± 0.0002 mAb22033 (50 mg/kg 34.3 ±2.1 0.124 ± 0.004 0.0069 ± 0.0002  0.0037 ± 0.0002* Telemeterednormotensive NPR1^(hu/hu) mice were randomized into five groups of equalbody weight and given a single subcutaneous injection of mAb22033 at thedoses listed in Table 1. An IgG4 antibody was used as isotype control.All values are mean ± SEM, n = 6-7 per group. Statistics - one way ANOVAwith Dunnett's; *p < .05 vs. IgG4 isotype control

Myocardial function (FIGS. 6 and 7A) was enhanced followingadministration of mAb22033, with a statistically significant reductionof end systolic volume noted in the 25 mg/kg dose group (FIG. 7A), andtrending increase in both fractional shortening (FIG. 7C) and ejectionfraction (FIG. 7B), most apparent in the 25 and 50 mg/kg groups.

The key findings are that the NPR1 agonist mAb, mAb22033 producedsignificant reductions in systolic and mean arterial blood pressuresthat lasted for up to 28 days. A compensatory increase in heart rate wasobserved in all groups initially, with a persistent minor increase inthe 50 mg/kg mAb22033 dose group compared to isotype control mAb.

Example 9: Effect of a Single Dose of an Agonist Anti-NPR1 MonoclonalAntibody on Systemic Blood Pressure in Hypertensive NPR1^(hu/hu) MiceExperimental Procedure

The objectives of this study were to assess the effects of a single doseof an NPR1 agonist antibody (mAb22033 or mAb22810) on systemic bloodpressure in telemetered angiotensin II-(Ang II)-induced hypertensiveNPR1^(hu/hu) mice. Male NPR1^(hu/hu) (n=36) mice aged ˜13 weeks wereimplanted with PA-C10 telemeters (DSI, St. Paul, Minn.) and allowed torecover for 7 days, prior to being assigned to group (Groups 1-6) (Table36).

TABLE 36 Summary of doses and dose groups Number Group Test or DoseLevel Dose Volume of Animals No. Control Article (mg/kg) (mL/kg) Males 1IgG4 isotype 25 5 5 control 2 mAb22033 1 5 4 3 mAb22033 5 5 5 4 mAb2203325 5 6 5 mAb22810 5 5 5 6 mAb22810 19 5 6

Animals were then implanted with osmotic minipumps (Alzet Micro-OsmoticPump; Model 1004; Lot 10335-14). Minipumps were filled with AngiotensinII acetate salt (Bachem; Lot #1066804) and set to a mean pumping rate of0.11 μL/hr for delivery of 1.5 mg/kg/day Ang II. Minipumps wereimplanted subcutaneously in the scapular region 3 days prior toinitiation of dosing. Animals were individually housed under standardconditions (Temperatures of 64° F. to 84° F. (18° C. to 29° C.);relative humidity of 30% to 70%) and a 12-hour light/12-hour dark cyclewas maintained. Food (Research Diets Standard pellet chow) and waterwere provided ad libitum.

The test proteins were administered subcutaneously to the appropriateanimals once on day 3 via subcutaneous injection. The dose volumes foreach animal were based on the most recent body weight measurement.

Systolic pressure, diastolic pressure, mean arterial pressure, and heartrate were collected for 10 seconds every minute for the duration of thetesting period. Urines were collected on day 14 and 20.

Results

A single dose of an NPR1 agonist mAb (mAb22033 or mAb22810)significantly reduced systemic blood pressures (FIGS. 8, 9 and 11) whenadministered to angiotensin-II-induced hypertensive NPR1^(hu/hu) mice.Mean 23-day blood pressures (Table 37) were all significantly lower inanimals that received either mAb22033 or mAb22810, compared to IgG4isotype control.

TABLE 37 23-day Mean Blood Pressures and Heart Rates Systolic DiastolicMean Arterial Heart Rate Dose Group: (mmHg) (mmHg) (mmHg) (BPM) IgG4isotype control (25 176 ± 2 147 ± 2 155 ± 2 507 ± 4 mg/kg) mAb22033 (1mg/kg) 163 ± 2**** 134 ± 2**** 147 ± 2 541 ± 4**** mAb22033 (5 mg/kg)153 ± 2**** 120 ± 1**** 132 ± 4**** 547 ± 7**** mAb22033 (25 mg/kg) 149± 2**** 121 ± 1**** 131 ± 4**** 544 ± 7**** mAb22810 (5 mg/kg) 162 ±2**** 128 ± 2**** 145 ± 2* 532 ± 5** mAb22810 (19 mg/kg) 159 ± 2**** 127± 2**** 139 ± 3*** 532 ± 4** All values are mean ± SEM, n = 3-6 pergroup. Statistics - two way ANOVA with Dunnett's; *p < .05 vs. IgG4isotype **p <.01 vs. IgG4 isotype control; ***p < .001 vs. IgG4 isotypecontrol; ****p < .0001 vs. IgG4 isotype control

TABLE 38 Urine and Serum Biomarkers Urine Urinary Urine Urinary PlasmaVolume cGMP Volume cGMP NTproANP (mL/day) - (mmol/day) - (mL/day) -(mmol/day) - (nmol/L) - Dose Group: Day 14 Day 14 Day 20 Day 20 Day 20IgG4 isotype control (25 2.2 ± 0.7 8.7 ± 2.5 5.2 ± 0.9 13.9 ± 3.1 0.7 ±0.1 mg/kg) mAb22033 (1 mg/kg) 3.0 ± 0.7 13.8 ± 2.3  6.6 ± 0.6 23.4 ± 2.80.8 ± 0.2 mAb22033 (5 mg/kg) 1.3 ± 0.5 11.0 ± 3.2  3.9 ± 0.3 20.8 ± 0.81.0 ± 0.3 mAb22033 (25 mg/kg) 2.1 ± 0.3 23.5 ± 5.3* 5.6 ± 0.8   44.9 ±7.8*** 1.5 ± 0.2 mAb22810 (5 mg/kg) 1.5 ± 0.4 8.3 ± 2.0 4.1 ± 0.8 19.2 ±5.3 1.3 ± 0.4 mAb22810 (19 mg/kg) 2.3 ± 0.3 13.6 ± 1.8  4.9 ± 0.7 18.8 ±1.9 1.0 ± 0.2 All values are mean ± SEM, n = 3-6 per group. Statistics -one way ANOVA with Dunnett's. *p <.05 vs. IgG4 isotype control; ***p <.001 vs. IgG4 isotype control

TABLE 39 Terminal Body and Organ Weights Terminal Heart Heart BodyWeight Heart Weight:Tibia Weight:Body Dose Group: (g) Weight (mg) LengthWeight IgG4 isotype control (25 29 ± 1 0.166 ± 0.011 0.0093 ± 0.00060.0057 ± 0.0003 mg/kg) mAb22033 (1 mg/kg) 32 ± 1 0.168 ± 0.005 0.0094 ±0.0004 0.0053 ± 0.0003 mAb22033 (5 mg/kg) 29 ± 2 0.149 ± 0.017 0.0085 ±0.0009 0.0051 ± 0.0005 mAb22033 (25 mg/kg) 32 ± 1 0.162 ± 0.008 0.0090 ±0.0004 0.0051 ± 0.0002 mAb22810 (5 mg/kg) 30 ± 1 0.150 ± 0.004 0.0084 ±0.0002 0.0050 ± 0.0001 mAb22810 (19 mg/kg) 30 ± 1 0.146 ± 0.015 0.0082 ±0.0008 0.0049 ± 0.0003 All values are mean ± SEM, n = 3-6 per group.Statistics - two way ANOVA with Dunnett's

Heart rate effects (FIG. 10) were significantly increased acutely, witha more modest increase chronically. Mean 23-day heart rates (Table 37)were significantly higher across all test article-administered groupscompared to isotype control animals. Urinary cGMP levels trended higherin most groups administered either mAb22033 or mAb22810, with animals inthe 25 mg/kg mAb22033 having significantly increased urine cGMP levelscompared to IgG isotype control animals at days 14 and 20 (Table 38). Noeffects on body weight or absolute or relative organ weights wereobserved (Table 39).

Example 10: Effect of Repeat Doses of an Agonist Anti-NPR1 MonoclonalAntibody on Systemic Blood Pressure in Hypertensive NPR1^(hu/hu) MiceExperimental Procedure

The objectives of this study were to assess the effects of repeat dosingof the NPR1 agonist antibody mAb22033 on systemic blood pressure intelemetered angiotensin II-(Ang II)-induced hypertensive NPR1^(hu/hu)mice. Male NPR1^(hu/hu) (n=30) mice aged ˜26 weeks were implanted withPA-C10 telemeters (DSI, St. Paul, Minn.) and allowed to recover for 7days, prior to being assigned to group (Groups 1-5) (Table 40).

TABLE 40 Summary of Doses and Dose Groups Number Group Test or DoseLevel Dose Volume of Animals No. Control Article (mg/kg/dose) (mL/kg)Males 1 IgG4 isotype 25 5 6 control 2 mAb22033  1 5 6 3 mAb22033  5 5 64 mAb22033 25 5 6 5 mAb22033  50* 5 6 *single dose

Animals were then implanted with osmotic minipumps (Alzet Micro-OsmoticPump; Model 1004; Lot 10335-14). Minipumps were filled with AngiotensinII acetate salt (Bachem; Lot #1066804) and set to a mean pumping rate of0.11 μL/hr for delivery of 1.5 mg/kg/day Ang II. Minipumps wereimplanted subcutaneously in the scapular region 7 days prior toinitiation of dosing. Animals were individually housed under standardconditions (Temperatures of 64° F. to 84° F. (18° C. to 29° C.);relative humidity of 30% to 70%) and a 12-hour light/12-hour dark cyclewas maintained. Food (Research Diets Standard pellet chow) and waterwere provided ad libitum.

Animals were stratified to group based off of systolic blood pressures.The test proteins were administered to the appropriate animals eitheronce on day 0 (Group 5) or twice weekly for three weeks (Groups 1-4)beginning on day 6 via subcutaneous injection. The dose volumes for eachanimal were based on the most recent body weight measurement.

Systolic pressure, diastolic pressure, mean arterial pressure, and heartrate were collected for 10 seconds every minute for the duration of thetesting period.

Results

Single or repeat doses of the NPR1 agonist mAb mAb22033 reduced systemicblood pressures (FIGS. 12, 13 and 15) back to near normotensive levelsin angiotensin-II-induced hypertensive NPR1^(hu/hu) mice. Repeated dosesof 1, 5 or 25 mg/kg mAb22033 dose-dependently reduced systolic bloodpressures, with peak reductions relative to individual group baselinesof 11, 19 and 39 mmHg, respectively after three weeks of twice weeklydosing. A single dose of 50 mg/kg reduced systolic blood pressure 31mmHg by day 7, with a gradual return to more hypertensive levelsthereafter. Mean 21-day blood pressures (Table 41) were allsignificantly lower in animals that received either single or repeateddoses of mAb22033, compared to IgG4 isotype control.

TABLE 41 21-day Mean Blood Pressures and Heart Rates Systolic DiastolicMean Arterial Heart Rate Dose Group: (mmHg) (mmHg) (mmHg) (BPM) IgG4isotype control 173.4 ± 1.3 138.6 ± 0.7 154.9 ± 1.2 538.2 ± 1.8 (25mg/kg) mAb22033 (1 mg/kg) 163.0 ± 1.2** 132.9 ± 1.0 146.8 ± 1.0** 532.5± 3.2 mAb22033 (5 mg/kg) 151.9 ± 0.9**** 124.7 ± 0.7**** 137.8 ± 0.8****524.8 ± 3.5** mAb22033 (25 mg/kg) 149.1 ± 2.2**** 127.0 ± 2.0*** 137.1 ±2.0**** 511.3 ± 2.9**** mAb22033 (50 mg/kg) * 156.2 ± 1.4**** 135.0 ±1.7 144.9 ± 1.5**** 552.4 ± 3.1** single dose* All values are mean ±SEM, n = 3-6 per group. Statistics - two way ANOVA with Dunnett's; **p <.01 vs. IgG4 isotype control; ***p < .001 vs. IgG4 isotype control;****p < .0001 vs. IgG4 isotype control

TABLE 42 Terminal Body and Organ Weights Terminal Heart Heart BodyWeight Heart Weight:Tibia Weight:Body Dose Group: (g) Weight (mg) LengthWeight IgG4 isotype control (25 30.9 ± 1.5 0.165 ± 0.006 0.0091 ±0.00032 0.0054 ± 0.0002 mg/kg) mAb22033 (1 mg/kg) 31.1 ± 1.1 0.151 ±0.007 0.0084 ± 0.00043 0.0049 ± 0.0002 mAb22033 (5 mg/kg) 29.6 ± 1.00.149 ± 0.004 0.0082 ± 0.00020 0.0050 ± 0.0001 mAb22033 (25 mg/kg) 32.4± 1.9 0.165 ± 0.012  0.009 ± 0.00067 0.0051 ± 0.0003 mAb22033 (50mg/kg) * single 31.0 ± 1.3 0.153 ± 0.006 0.0085 ± 0.00034 0.0050 ±0.0003 dose* All values are mean ± SEM, n = 3-6 per group. Statistics -one way ANOVA with Dunnett's

TABLE 43 Urine and Serum Markers Serum Urine Urinary NTproANP VolumecGMP Serum NTproANP (nmol/L) - Dose Group: (mL/day) (mmol/day)(nmol/L) - Day 20 Day 29 IgG4 isotype control (25 2.74 ± 0.74 30.5 ±6.3  0.58 ± 0.05 0.57 ± 0.09 mg/kg) mAb22033 (1 mg/kg) 3.08 ± 0.51 61.6± 20.4 0.75 ± 0.03 0.51 ± 0.05 mAb22033 (5 mg/kg) 2.48 ± 0.68 36.6 ±10.4 0.45 ± .013 0.36 ± 0.08 mAb22033 (25 mg/kg)  3.2 ± 0.80 *106.3 ±25.4*  0.59 ± 0.13 0.63 ± 0.14 mAb22033 (50 mg/kg) * single 3.27 ± 0.8952.8 ± 21.5 0.56 ± 0.18 1.27 ± 0.43 dose* All values are mean ± SEM, n =3-6 per group. Statistics - one way ANOVA with Dunnett's. *p < .05 vs.IgG4 isotype control

Acute effects of mAb22033 were analyzed, with pressure reductions of10-20 mmHg within 24 hours of the first dose. Heart rate effects (FIG.14) were variable, with higher and lower values being observed over the21-day dosing period. Mean 21-day heart rates (Table 41) weresignificantly lower in the 5 and 25 mg/kg repeat dose groups, andtrended higher in the single 50 mg/kg dose group. Urinary cGMP levelswere significantly increased in the 25 mg/kg repeat dose group, with allother groups showing trending, non-statistically significant increasescompared to IgG4 isotype control mAb dosed animals (Table 43). Noeffects on body weight, absolute or relative organ weights, standardserum or urine chemistries or cardiac function were observed (Table 42and FIGS. 16-17).

Example 11: Effect of Anti-NPR1 Agonist Antibodies on Body Weight,Metabolic Rate and Glucose Homeostasis in Diet-Induced Obese (DIO) MiceExperiment 1

In this experiment, the effect of mAb22810 NPR1 agonist mAb on bodyweight, metabolic rate and glucose homeostasis in diet-induced obese(DIO) mice was tested.

Thirty, male NPR1^(hu/hu) mice were placed on a 60% high-fat diet for 10weeks, then randomized into three groups (n=10 per group): Isotypecontrol (human IgG4) antibody, NPR1 agonist antibody mAb22810 orhFc.FGF21. FGF21 is a molecule that has been proven to improve glucosetolerance, increase energy expenditure and lower body weight in obesemice models (Véniant M M, Endocrinology, 2012, PMID: 22798348). It wasused as a positive control in this study for the endpoints. Treatmentswere administered in saline vehicle by subcutaneous injection (S.C.)either weekly (for the control antibody and mAb22810) or twice weekly(for hFc.FGF21). Table 44 lists the groups, number of animals and dosesin the study.

TABLE 44 Summary of treatment groups Group Treatment Dose DosingSchedule Mice 1 Control mAb 25 mg/kg 1x/week, S.C. 10 2 mAb22810 25mg/kg 1x/week, S.C. 10 3 hFc.FGF21 3.2 mg/kg  2x/week, S.C. 10

Individual body weights were recorded prior to dosing and twice per weekthereafter. During the second week of the study, mice were put intometabolic cages to assess energy expenditure. Oral glucose tolerance wasassessed during the third week of the study and body composition wasmeasured after six weeks of treatment by EchoMRI.

FIG. 18A shows changes in body weight following administration ofmAb22810 NPR1 agonist mAb, hFc.FGF21 or an isotype control mAb. FIG. 18Band FIG. 18C show total fat and total lean mass respectively after sixweeks of treatment as measured by EchoMRI. The key findings are thatwhile the hFc.FGF21 molecule produced significant reductions in bodyweight and adiposity over the treatment period, the mAb22810 NPR1agonist antibody did not.

FIGS. 19A-C show changes in VO₂ (A), VCO₂ (8) or Energy Expenditure (C)broken down as the average of each day/night cycle after one week oftreatment with either mAb22810 NPR1 agonist mAb, hFc.FGF21 or an isotypecontrol mAb. The key findings are that while the hFc.FGF21 moleculeproduced significant increases in VO₂, VCO₂ and energy expenditure overthe treatment period, the mAb22810 NPR1 agonist antibody did not.

FIG. 20A shows changes in glucose tolerance as measured by an oralglucose tolerance test (2 g/kg glucose) after two weeks of treatmentwith either mAb22810 NPR1 agonist mAb, hFc.FGF21 or an isotype controlmAb. FIG. 20B shows glucose levels following an overnight fast asrecorded at the start of the study in A. The key findings are that boththe mAb22810 and hFc.FGF21 molecules produced significant improvementsin glucose tolerance after two weeks. Furthermore, the improvement inglucose tolerance by mAb22810 was independent of changes in body weightor energy expenditure (as shown in FIGS. 18 and 19).

Experiment 2

This experiment describes the effect of mAb22033 NPR1 agonist mAb onbody weight, metabolic rate and glucose homeostasis in diet-inducedobese (DIO) mice.

Thirty, male NPR1^(hu/hu) mice were placed on a 60% high-fat diet for 10weeks, then randomized into three groups (n=10 per group): Isotypecontrol (human IgG4) antibody, NPR1 agonist antibody mAb22033 orhFc.FGF21 as a positive control. Treatments were administered in salinevehicle by subcutaneous injection (S.C.) either weekly (for the controlantibody and mAb22033) or twice weekly (for hFc.FGF21). Table 45 liststhe groups, number of animals and doses in the study.

TABLE 45 Summary of treatment groups Group Treatment Dose DosingSchedule Mice 1 Control mAb 25 mg/kg lx/week, S.C. 10 2 mAb22033 25mg/kg lx/week, S.C. 10 3 hFc.FGF21 3.2 mg/kg  2x/week, S.C. 10

Individual body weights were recorded prior to dosing and twice per weekthereafter. During the second week of the study, mice were put intometabolic cages to assess energy expenditure. Oral glucose tolerance wasassessed during the fourth week of the study and body composition wasmeasured after six weeks of treatment by EchoMRI.

FIG. 21A shows changes in body weight following administration ofmAb22033 NPR1 agonist mAb, hFc.FGF21 or an isotype control mAb. FIG. 21Band FIG. 21C show total fat and total lean mass respectively after sixweeks of treatment as measured by EchoMRI. The key findings are thatwhile the hFc.FGF21 molecule produced significant reductions in bodyweight and adiposity over the treatment period, the mAb22033 NPR1agonist antibody did not.

FIGS. 22A-C show changes in VO₂ (A), VCO₂ (8) or Energy Expenditure (C)broken down as the average of each day/night cycle after one week oftreatment with either mAb22033 NPR1 agonist mAb, hFc.FGF21 or an isotypecontrol mAb. The key findings are that while the hFc.FGF21 moleculeproduced significant increases in VO₂, VCO₂ and energy expenditure overthe treatment period, the mAb22033 NPR1 agonist antibody did not.

FIG. 23A shows changes in glucose tolerance as measured by an oralglucose tolerance test (2 g/kg glucose) after four weeks of treatmentwith either mAb22033 NPR1 agonist mAb, hFc.FGF21 or an isotype controlmAb. FIG. 23B shows glucose levels following an overnight fast asrecorded at the start of the study in A. The key finding is that boththe mAb22033 and hFc.FGF21 molecules produced significant improvementsin glucose tolerance after two weeks. Furthermore, the improvement inglucose tolerance by mAb22033 was independent of changes in body weightor energy expenditure (as shown in FIGS. 21 and 22).

Example 12: HDX Epitope Mapping

In order to determine the epitopes of human NPR1 recognized by anti-NPR1antibodies, hydrogen-deuterium exchange (HDX) studies were carried outfor mAb22033 and mAb22810, respectively. Prior in-house experiments showthat the binding of NPR1 to mAb22810 requires the presence of ANPpeptide. Hence, in addition to conventional HDX experiment usingNPR1/mAb22033 complex, HDX experiments were also performed forNPR1/ANP/mAb22033 and NPR1/ANP/mAb22810 complexes.

For this study, anti-NPR1 antibodies (mAb22033 and mAb22810) werecovalently attached to N-hydroxysuccinimide (NHS) agarose beads (GELifescience, cat #17-0906-01) according to manufacturer's protocol. CHOcell-expressed recombinant human NPR1 protein comprising the ecto domainof human NPR1 protein (Uniprot accession # P16066) with a C-terminalmyc-myc-hexahistidine tag (SEQ ID NO: 194). ANP peptide was purchasedfrom TOCRIS (cat #1906).

The deuteration buffer was prepared in D₂O containing 137 mM NaCl, 2.7mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO₄ (pD=7.4). For either “antigen-on”or “complex-on” experiments, 30 μL antibody bead slurry (equiv. 15 μLbeads) was mixed with hNPR1.mmh or hNPR1.mmh/ANP complex. The mixturewas incubated at room temperature with gentle rotation. The deuterationwas quenched along with the elution of hNPR1.mmh from the antibody beadsusing 0.075% ice-cold TFA. The quenched sample was immediately injectedinto a Waters HDX Manager for online pepsin digestion (Waters EnzymateBEH pepsin column, 2.1×30 mm). The digested peptides were trapped ontoan ACQUITY UPLC BEH C18 1.7 μm, 2.1×5 mm VanGuard pre-column at 0° C.and eluted to an ACQUITY UPLC BEH C18 1.7 μm, 2.1×50 mm column using an8-minute gradient separation of 1%-30% B (mobile phase A: 0.1% formicacid in water, mobile phase B: 0.1% formic acid in acetonitrile). Themass spectrometer was set at cone voltage of 37 V, scan time of 0.5 s,and mass/charge range of 50-1700 Th.

To map the hNPR.mmh binding epitope recognized by mAb22033, two sets ofH/D exchange experiments were carried out. The first experiment used an“antigen-on” format (HDX of antigen alone followed by binding toantibody beads). For the “antigen-on” experiment, hNPR1.mmh wasdeuterated for 3 and 8 minutes (in two separate sub-experiments) inphosphate buffer prepared in D₂O (PBS-D, pD=7.4) at room temperature.Deuterated hNPR1.mmh was subsequently added to the PBS-D-washed mAb22033beads for a 2-minute incubation at room temperature, resulting in atotal deuteration time of 5 minutes and 10 minutes, respectively. Thebound hNPR.mmh was then eluted from the beads using an ice-cold 0.075%aqueous trifluoroacetic acid (TFA) solution. The eluted hNPR.mmh wasimmediately injected into a Waters HDX manager system for online pepsindigestion followed by peptic peptide mass measurement.

The second experiment is referred to as the “complex-on” format (HDX ofcomplexed antigen/antibody beads). For this experiment, hNPR.mmh wasfirst bound to the mAb22033 beads in regular PBS (pH=7.4) for 2 minutes.The complex was then incubated in PBS-D (pD=7.4) for 5 or 10 minutes (inseparate sub-experiments) for deuteration. The following steps (elution,injection, pepsin digestion, and MS analysis) were carried out asdescribed in the preceding “antigen-on” procedure.

For peptide identification and deuterium uptake measurements, LC-MSEdata from un-deuterated hNPR1.mmh were first processed and searchedagainst the database including hNPR1.mmh via Waters ProteinLynx GlobalServer (PLGS) software. The identified peptides were imported to DynamXsoftware and filtered by two criteria: 1) minimum products per aminoacid: 0.3, and 2) replication file threshold: 2. DynamX software wasthen able to determine the deuterium uptake of each peptide from the“antigen-on”, “complex-on” experiments based on each peptide's retentiontime and mass accuracy (<30 ppm) across two time points. All identifiedpeptides were inspected and screened manually to minimize false positivehits.

The centroid values or average mass-to-charge ratios (m/z) of all thedetected peptides were calculated and compared between the “antigen-on”and “complex-on” experiments at two time points. Peptides exhibitingincreased mass after the “antigen-on” deuteration compared to the“complex-on” deuteration include amino acids protected from deuteriumexchange as a result of antibody binding and therefore reveal bindingepitope regions.

For NPR1/mAb22033 HDX experiment, a total of 101 peptides from hNPR1.mmhwere identified, representing 74% sequence coverage. Among thesepeptides, ten peptides covering amino acids spanning from 29-50 and328-347 had significant increased mass after the “antigen-on”deuteration compared to the “complex-on” deuteration as listed in Table46.

TABLE 46 The effect on H/D exchange of mAb22033 binding to hNPR1.mmh asmeasured by MH⁺ values of peptic peptides NPR1- 5 min Deuteration 10 minDeuteration mmH Antigen- Complex- Antigen- Complex- Residues on on Δ onon Δ 29-50 2426.346 2424.541 1.80 2426.649 2424.925 1.72 32-50 2128.2002126.776 1.42 2128.488 2127.088 1.40 46-54 950.481 950.333 0.15 950.549950.527 0.02 328-335 925.039 925.027 0.01 925.065 925.030 0.03 328-3472084.447 2081.863 2.58 2084.306 2081.925 2.38 329-347 1921.240 1918.3352.91 1921.305 1918.614 2.69 331-347 1679.173 1677.107 2.07 1679.1061677.176 1.93 332-347 1607.797 1605.754 2.04 1607.799 1605.842 1.96334-347 1407.379 1405.529 1.85 1407.335 1405.612 1.72 335-347 1278.1961276.411 1.78 1278.225 1276.463 1.76 336-347 1176.174 1174.913 1.261176.181 1174.926 1.26 337-347 1063.079 1061.760 1.32 1063.014 1061.7451.27

Since two peptides, amino acids 46-54 and 328-335 did not show deuteriumuptake difference between the “antigen-on” and “complex-on” procedures,the regions of protection from deuterium exchange in the 29-50 and328-347 peptides are reduced to residues 29-45 and 336-347. Therefore,two segments including amino acids 29-45 and 336-347 are identified asthe epitope for antibody mAb22033 binding to the hNPR1.mmh protein.

For NPR1/ANP/mAb22033 HDX experiment, a total of 95 peptides fromhNPR.mmh were identified, representing 68% sequence coverage. Amongthese peptides, nine peptides covering amino acids spanning from 29-50and 331-347 had significant increased mass after the “antigen-on”deuteration compared to the “complex-on” deuteration as listed in Table47.

TABLE 47 The effect on H/D exchange of mAb22033 binding to hNPR1.mmh/ANPas measured by MH⁺ values of peptic peptides NPR1- 5 mm Deuteration 10mm Deuteration mmH Antigen- Complex- Antigen- Complex- Residues on on Δon on Δ 29-50 2425.746 2424.277 1.47 2426.686 2425.079 1.61 32-502127.514 2126.429 1.08 2128.333 2127.126 1.21 35-50 1828.391 1827.3311.06 1829.026 1827.974 1.05 46-54 950.149 950.080 0.07 950.312 950.323−0.01 331-347 1679.030 1677.150 1.88 1679.016 1677.216 1.80 332-3471607.700 1605.825 1.88 1607.738 1605.943 1.79 334-347 1407.282 1405.5581.72 1407.271 1405.715 1.56 335-347 1278.028 1276.463 1.56 1278.0871276.658 1.43 336-347 1176.211 1175.009 1.20 1176.186 1175.009 1.18337-347 1063.036 1061.743 1.29 1063.019 1061.807 1.21

Since another peptide, amino acids 46-54, did not show deuterium uptakedifference between the “antigen-on” and “complex-on” procedures, theregion of protection from deuterium exchange in the 29-50 peptide isreduced to residues 29-45. Therefore, two segments including amino acids29-45 and 331-347 are identified as the epitope for antibody mAb22033binding to the hNPR1.mmh/ANP protein complex.

For NPR1/ANP/mAb22810 HDX experiment, a total of 93 peptides fromhNPR.mmh were identified, representing 70% sequence coverage. Amongthese peptides, ten peptides covering amino acids spanning from 29-50,70-81 and 331-347 had significant increased mass after the “antigen-on”deuteration compared to the “complex-on” deuteration as listed in Table48.

TABLE 48 The effect on H/D exchange of mAb22810 binding to hNPR1.mmh/ANPas measured by MH⁺ values of peptic peptides NPR1- 5 mm Deuteration 9910 mm Deuteration mmH Antigen- Complex- Antigen- Complex- Residues on onΔ on on Δ 29-50 2426.295 2425.324 0.97 2426.927 2426.077 0.85 32-502128.086 2127.361 0.72 2128.584 2128.016 0.57 33-50 2056.507 2056.0830.42 2056.723 2056.405 0.32 35-50 1828.973 1828.597 0.38 1829.2781828.952 0.33 46-54 950.063 950.090 −0.03 950.267 950.087 0.18 70-811456.036 1455.661 0.37 1456.127 1455.699 0.43 72-81 1241.898 1241.4960.40 1241.969 1241.536 0.43 331-347 1678.712 1678.316 0.40 1678.8881678.589 0.30 332-347 1607.457 1607.060 0.40 1607.617 1607.323 0.29334-347 1407.018 1406.698 0.32 1407.217 1406.970 0.25 335-347 1277.8161277.550 0.27 1278.075 1277.824 0.25 336-347 1175.926 1175.894 0.031176.094 1176.051 0.04 337-347 1062.703 1062.646 0.06 1062.844 1062.7670.08

Since three peptides, amino acids 46-54, 336-347, 337-347 did not showdeuterium uptake difference between the “antigen-on” and “complex-on”procedures, the regions of protection from deuterium exchange in the29-50, and 331-347 peptides are reduced to residues 29-45 and 331-335.Therefore, three segments including amino acids 29-45, 70-81 and 331-335are identified as the epitope for antibody mAb22810 binding to thehNPR1.mmh/ANP protein complex.

Example 13: Intravitreal Injection of NPR1 Antibody in Humanized NPR1Mice Lowers Intraocular Pressure

This Example describes the effect of an intravitreal injection (IVT) ofexemplary human NPR1 antibody mAb22033 on intraocular pressure (IOP) inhumanized NPR1 mice.

Methods:

Humanized NPR1 mice (NPR1^(hu/hu)) were generated with VelociGenetechnology (Regeneron). A single IVT injection of 40 μg mAb22033orcontrol Ab was performed in humanized NPR1 or wild-type (VVT) mice. IOPwas measured daily for four days after injection. In a secondexperiment, to examine the dose response, 40, 12.6, or 4 μg mAb22033or40 μg control Ab was injected intravitreally, and IOP was monitoreddaily. In another experiment, to test the effect of long-term deliveryAb, AAV2 vectors expressing NPR1 antibody or eGFP were injected, IOP wasfollowed up for 7 weeks.

Results:

IVT of 40 μg mAb22033 into NPR1^(hu/hu) mice significantly reduced IOPfrom Day 1 to 3 compared to control antibody. Average IOP change was 5mmHg. However, in WT mice there was no IOP lowering effect. Doseresponse study showed that 40 or 12.6 μg IVT of mAb22033 had similar IOPlowering effect, while the effect of 40 μg mAb22033 lasted longer than12.6 μg. IVT of 4 μg NPR1 antibody did not reduce IOP. No IOP effect wasfound after IVT AAV2-GFP or AAV2-NPR1 antibody at all experimental timepoints. This could be due to the low expression of NPR1 antibody i.e.only 10 ng in the whole eye lysate was detected.

Conclusion: IVT administration of human NPR1 antibody (mAb22033) inhumanized NPR1 mice reduced IOP significantly, thus demonstrating thepotential of agonist anti-NPR1 antibodies for lowering IOP in glaucomadisease.

Example 14: Structural Analysis of the Antibody-NPR1 Complex by ElectronMicroscopy Methods

Size Exclusion Chromatography with Multi Angle Light Scattering(SEC-MALS) Titrations

Several titration series of human NPR1 extracellular domain with aC-terminal myc-myc-6×His tag (hNPR1-mmh; SEQ ID NO: 194) complexed withvarious antibodies at different molar ratios were prepared. Theantibodies tested were: mAb22033, REGN5308 (Fab fragment of mAb22033),mAb22810, and REGN5314 (Fab fragment of mAb22810). All titration serieswere done both with and without Atrial Natriuretic factor (ANP, Tocris)at a 2-fold molar excess relative to hNPR1-mmh. After overnightincubation in PBS at 4° C., the complexes were injected into theSEC-MALS system, which consists of a Superdex 200 Increase 10/300 GIcolumn on an AKTA micro system (GE Healthcare Life Sciences), followedby a miniDAWN Treos and Optilab T-rEX (Wyatt Technology Corporation).Since phosphate buffers are incompatible with electron microscopynegative staining, the SEC column was equilibrated in 50 mM Tris pH 7.5,150 mM NaCl running buffer, and all of the larger scale complexesprepared below were in this buffer. Size exclusion chromatography datawas evaluated using Unicorn (Version 5.20 General Electric Company), andthe MALS data was evaluated using ASTRA (Version 7.0.0.69 WyattTechnology).

Negative Stain Electron Microscopy Sample Preparation

Complexes of hNPR1 were prepared on a larger scale to use innegative-stain electron microscopy. Five samples were prepared asfollows: Sample 1=hNPR1-mmh (SEQ ID NO: 194) alone; Sample 2=hNPR1-Fc(SEQ ID NO: 197) alone; Sample 3=hNPR1-mmh+ANP, 1:2 molar ratio; Sample4=hNPR1-mmh+REGN5308, 1:1.5 molar ratio; Sample5=hNPR1-mmh+ANP+REGN5308, 1:2:1.5 molar ratio. Samples 1, 3, 4, and 5were purified by size exclusion chromatography in the same manner as theSEC-MALS experiments. Peak fractions were collected, frozen at −80 C,and sent to NanoImaging Services, Inc. for EM analysis. Sample 2 wastaken directly from a 2.62 mg/ml stock solution in PBS, buffer exchangedto 50 mM Tris, pH 7.5+150 mM NaCl, diluted to 1.5 mg/ml, frozen at −80C, and sent with the other samples.

Negative Stain Electron Microscopy Data Collection and Processing

The five protein samples were prepared negative stain EM grids in thestandard manner using uranyl formate (NanoImaging Services). Gridscontained a thin layer of continuous carbon placed over a C-flat holeycarbon grid. TEM images were collected at room temperature on a TecnaiT12 electron microscope (FEI/Thermo Fisher) operating at 120 keV with anFEI Eagle 4 k×4 k CCD camera. Images were collected at a variety ofnominal magnifications, primarily 67,000× and 110,000×. The collectedimages were further processed in-house.

The NanoImaging micrographs by eye were inspected by eye, and imagesthat had poor stain contrast were removed, about ⅕ of the total images.The remaining images were separated by magnification; the 110,000×images were not as useful for further analysis since they had fewerparticles per image. Therefore, all subsequent processing steps weredone using the 67,000× images. All images were CTF-corrected usingCTFFIND4.

EM Particle Picking and 2D Class Averaging

The particle distributions for all five negative stain samples werequite good, with uniform particle size, very little clumping, and a goodparticle density. In Samples 4 and 5, particles were picked usingRelion, with autopicking templates derived from an initial round ofmanual picking and 2D class averaging. For Sample 4 (hNPR1+REGN5308),19184 good particles were selected from a total of 75 micrographs. ForSample 5 (hNPR1+ANP+REGN5308), 20318 good particles were initiallyselected from a total of 88 micrographs. Initial 2D class averagingusing Relion revealed substantial heterogeneity in both datasets, with asubstantial number of classes showing REGN5308 Fab only, or NPR1 only.

The 2D class averaging for Sample 5 was refined further by eliminatingparticles corresponding to Fab only or NPR1 only. The remaining 9219particles were used to calculate new 2D class averages, which showed abetter distribution of views for the NPR1+REGN5308 complex, and revealedthat a minority of the complexes contained only one Fab bound to theNPR1 dimer, while the majority of the complexes contained two Fabs.Removing the one-Fab complexes reduced the particle set further to 6728particles.

3D Image Reconstruction from Negative Stain EM Data

An initial 3D model of the NPR1-ANP-REGN5308 complex was constructed inRelion, using the stochastic gradient descent “3D initial model”procedure, with resolution limited to 40 Å. This model was then furtherlow-pass filtered to 60 Å and used as a reference for 3D classificationin Relion of the 6728 particles mentioned above, with expectation stepresolution limited to 25 Å. The best 3D class was then further refinedin Relion until convergence, with a final resolution of 22 Å as measuredby the “gold standard” FSC. We did not impose 2-fold symmetry during the3D classification or refinement. The density map resulting from the 3Dreconstruction clearly showed two Fabs bound on one side of a squareparticle consistent with the crystal structure of ANP-bound NPR1 (PDBcode 1T34).

Cryo-Electron Microscopy Sample Preparation and Data Collection

A sample of the NPR1-ANP-REGN5308 complex was prepared for cryo-electronmicroscopy (cryoEM) in the same manner as the negative stain EM sampledescribed above. The final complex concentration was 0.8 mg/ml in 50 mMTris, pH 7.5, 150 mM NaCl. CryoEM samples were prepared with standardtechniques, using UltrAuFoil grids (Quantifoil Micro Tools GmbH) and aVitrobot (FEI/Thermo Fisher). Data collection was done on a Titan Krioselectron microscope operating at 300 kV (FEI/Thermo Fisher) using a K2direct electron detector in counting mode, and GIF energy filter (Gatan,Inc.). 1409 movies were collected at a magnification of 130,000× (1.04Å/pixel) with a defocus range of −0.5 to −1.5 microns, and a total doseof 45.44 e⁻/Å². Data collection was controlled by Leginon software.

CryoEM Data Processing and Structure Determination

All cryoEM movies were motion-corrected, dose-weighted, and CTFcorrected using the cisTEM package. Images were then manually inspectedto remove those with thick ice, poor CTF parameters (fit resolutionworse than 6 Å), no particles, contamination, etc. 1172 images remainedafter this filter, which were then used for non-templated particlepicking in cisTEM, yielding 872915 particle positions. After 2Dclassification to remove the bad particles, 686709 particles wereincluded in 3D auto-refinement using cisTEM, with a starting 3Dreference volume generated ab initio. The 3D refinement converged to asingle solution with a resolution of 2.8 Å estimated from the FourierShell Correlation curve.

This 3D map was then used for structure refinement, starting from amodel built into the negative stain EM 3D map mentioned above. The N-and C-terminal domains of both NPR1 molecules were real-space refined asrigid bodies into the EM map, and then manually rebuilt in the fewplaces where the model did not match the EM density. The REGN5308homology model was manually placed into the EM density; carefulinspection of the CDR regions allowed us to determine the orientation ofthe heavy vs. light chains. The CDR regions of the model neededextensive rebuilding to match the EM density. Finally, real-spacepositional refinement using Phenix produced the current structural modelof the complex.

Results/Discussion

Size Exclusion Chromatography with Multi Angle Light Scattering(SEC-MALS) Titrations

In interactions of hNPR1-mmh with mAb22033, hNPR1-mmh by itself behavesas a dimer with a molecular weight of approximately 110 kDa in thepresence and absence of ANP, with a slight increase in molecular weightupon ANP binding to NPR1. No free monomer peak was observed forhNPR1-mmh by itself. Titrations with mAb22033 in the absence of ANPrevealed two main species of the complex: a species with molecularweight equal to one IgG bound to one NPR1 dimer, and a species withmolecular weight equal to one IgG bound to two NPR1 dimers. However,NPR1 plus mAb22033 in the presence of ANP formed higher molecular weightspecies that could represent “paper-doll” polymers of NPR1 and IgG.

The system was subsequently simplified by considering REGN5308, the Fabfragment of mAb22033. Complexes of hNPR1-mmh and REGN5308 show a verydifferent SEC profile with bound ANP compared to the same complexeswithout ANP. The NPR1-REGN5308 complex has a molecular weight ofapproximately 155 kDa, consistent with one Fab bound per NPR1 dimer. Inthe presence of ANP, the NPR1-ANP-REGN5308 complex molecular weightincreases by ˜50 kDa, consistent with two Fabs bound per NPR1 dimer. Wepropose that the previously-described conformational change in NPR1 uponANP binding (Ogawa, H et. al, 2004) permits the second Fab to bind, andthat the 2 Fab+2 NPR1+ANP complex is necessary for the growth of thepaper-doll polymers observed with the full IgG mAb22033 (see below forfurther discussion).

Titrations with hNPR1-mmh and mAb22810 were also carried out. SEC-MALSanalysis showed that a small fraction of the mAb22810 sample wasdimeric, with a molecular weight of approximately 315 kDa compared tothe standard IgG molecular weight of 150 kDa. SEC-MALS titrations withthe NPR1-mAb22810-ANP complex showed a heterogeneous mixture of speciesin the 430-700 kDa range; this profile was too complex to be reliablyinterpreted, perhaps due to the IgG dimer impurity in the mAb22810protein. SEC-MALS titrations with REGN5314, the Fab fragment ofmAb22810, showed that REGN5314 binding to NPR1 in the absence of ANP istoo weak or transient to produce a complex species that can be isolatedby SEC. When ANP is present, a single NPR1-REGN5314-ANP complex isformed with a molecular weight of approximately 170 kDa, consistent withone Fab bound per NPR1 dimer.

Negative Stain 2D Class Averages

The hNPR1+REGN5308 and the hNPR1+ANP+REGN5308 complexes were furtheranalyzed by negative stain electron microscopy. 2D classification andaveraging of the complex particles revealed substantial heterogeneity inthe samples. A large fraction of the particles on the EM grids could beclassified as hNPR1 only, or as REGN5308 Fab only. Since the proteinsamples submitted for imaging were purified, homogeneous complexes, itappears that these complexes are dissociating into their componentsduring the process of negative stain grid preparation. However, a goodportion of the complexes remained intact and can be used for analysis.

The negative stain 2D class averages of hNPR1+REGN5308 show a “onearmed” complex, in which only a single Fab can be seen binding to thehNPR1 dimer, consistent with the SEC-MALS results. In contrast, thenegative stain 2D class averages of hNPR1+ANP+REGN5308 show a “twoarmed” complex, with two Fabs bound to the dimer. Different averagesrepresent different projection views of the real three-dimensionalcomplex. In one class average, the hNPR1 dimer is seen in a side view,with two lobes of density: one corresponding to the N-terminal domainsof the two monomers superimposed on each other, and the other lobecorresponding to the two superimposed C-terminal domains. In thisorientation, the two bound Fabs look like “rabbit ears” sitting on topof the NPR1 density. In a different view, all four domains of hNPR1 canbe seen as four blobs of density forming a square, with the two REGN5308Fabs above the square, crossing over each other to make an inverted V.The hNPR1+ANP+REGN5308 class averages also showed “one armed” complexes,but these are likely due to the same complex dissociation that producedfree Fab and free NPR1.

2D class averages calculated from the hNPR1+ANP+REGN5308 cryoEM datashow different views of the complex compared to the negative stain data;in particular, the “rabbit ears” orientation is not present. However,other cryoEM 2D class averages can be matched closely to thecorresponding averages in the negative stain data. We do not see as muchevidence of complex dissociation in the cryoEM data, probably becausethe starting sample is more homogeneous, and also because thecryo-freezing conditions better preserve the native conformation of thecomplex in solution.

3D Image Reconstruction

A three-dimensional map of the cryoEM density for hNPR1+ANP+REGN5308 wasconstructed using the 2D class averages as a starting point. Thisreconstruction procedure does not require any prior information aboutthe expected shape or size of the complex, beyond a rough estimate ofthe complex diameter, so the resulting cryoEM map is unbiased by ourexpectations of how the antibody-target complex should be formed. Theknown crystal structure of hNPR1+ANP was then placed and refined intothis EM density map, along with models of the REGN5308 Fabs generated byhomology to known Fab structures. The NPR1 and Fab structures wereplaced in their approximate locations by hand, and then refined as rigidbodies into the correct locations using Phenix. The resolution of thecryoEM maps is sufficient to permit manual rebuilding of the residues atthe NPR1:antibody contact interface, particularly the residues of theREGN5308 complementarity-determining regions (CDRs), which cannot beaccurately modeled by homology. The current structural model has placedall antibody CDR residues as well as the NPR1 residues in contact withthem. More distant regions of the model (NPR1 C-terminal domains, andthe constant domains of the antibody Fabs) have been modeled through acombination of the current cryoEM map and previously-determined X-raycrystal structure information for NPR1 (PDB code 1T34) and isolatedantibody structures.

mAb22033 Epitope on NPR1:

Inspection of the hNPR1+ANP+REGN5308 structure reveals which residues ofNPR1 are contacted by the REGN5308 Fab (and by extension, the parentalIgG mAb22033). This epitope is composed of four separate stretches ofamino acids in NPR1 which combine to form a continuous surface in threedimensions: residues 2-4, 41-45, 47, 73-79, 332, 336-344, and 347(numbered according to SEQ ID NO: 194). Earlier hydrogen/deuteriumexchange (HDX) mass spectrometry experiments identified some of theseresidues as significant (see Example 12), but the cryoEM structureprovides finer detail of the epitope.

Structural Mechanism of Action for mAb22033

The two Fabs in this model of the NPR1-antibody complex come within ˜10Å of each other at a point near the “elbow” between Fab variable domainsand Fab constant domains. The C termini of these two Fabs are muchfarther apart, approximately 100 Å, and therefore could not be the twoarms of a single IgG molecule. The Fabs do not come particularly closeto the modeled ANP peptide (roughly 30 Å distance at closest approach),and it does not seem that there is any direct interaction between Faband ANP.

If we assume a fixed position and relative orientation of the Fabrelative to its binding site on the N-terminal domain of NPR1, then anexplanation of the ANP-dependent binding of the REGN5308 Fab emerges.NPR1 has been shown to undergo a conformational change upon ANP binding,in which one NPR1 monomer rotates relative to the other while remainingdimerized. Applying this rotation to one-half of the 2 NPR1+2 Fabcomplex produces a model in which the NPR1 dimer resembles the crystalstructure of ANP-free NPR1. However, one of the REGN5308 Fabs has nowrotated into a position where it sterically clashes with the other Fab,a physically impossible situation. This steric interference is thereason why only one REGN5308 Fab can bind to the NPR1 dimer in theabsence of ANP: both antibody binding sites on the two monomers areequally accessible but binding of the first Fab blocks binding of thesecond Fab.

If we consider this effect from the other side, the binding of two Fabsto an ANP-containing NPR1 dimer will prevent this complex from relaxingback to the ANP-free conformation, so long as both Fabs are bound. Atequilibrium, this would cause a larger fraction of the NPR1 molecules tobe in an active state capable of downstream signaling—if we assume thatthe effects we see here are preserved on the surface of a cell. Anotherpossible effect of antibody binding is the formation of oligomericclusters of antibody with NPR1+ANP as described above. This effect isonly possible if each NPR1 dimer can bind two Fab arms from two separateIgG molecules; in the absence of ANP, only one Fab arm can bind to eachNPR1 dimer, and the complex formation stops with a much smaller speciescontaining at most one IgG with an NPR1 dimer bound to each Fab arm.Both of these effects, receptor clustering and prolongation of receptoractive state, could explain the activating effect of mAb22033 on theNPR1 receptor.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. An isolated antibody or antigen-binding fragmentthereof that binds specifically to natriuretic peptide receptor 1 (NPR1)protein, wherein the antibody or antigen-binding fragment thereofinteracts with one or more amino acids contained within theextracellular domain of NPR1 (amino acids 29-347 of SEQ ID NO: 194), asdetermined by hydrogen/deuterium exchange, and wherein the antibody orantigen-binding fragment thereof: (i) binds to cells expressing humanNPR1 in the presence or absence of atrial natriuretic peptide (ANP);and/or (ii) binds to NPR1 and activates NPR1.
 2. The isolated antibodyor antigen-binding fragment thereof of claim 1, wherein the antibody orantigen-binding fragment thereof interacts with an amino acid sequenceselected from the group consisting of (a) amino acids 29 to 45 of SEQ IDNO: 194; (b) amino acids 331 to 347 of SEQ ID NO: 194; (c) amino acids336 to 347 of SEQ ID NO: 194; (d) amino acids 331 to 335 of SEQ ID NO:194; and (e) amino acids 70 to 81 of SEQ ID NO:
 194. 3. The isolatedantibody or antigen-binding fragment thereof of claim 2, wherein theantibody or antigen-binding fragment thereof interacts with an aminoacid sequence selected from the group consisting of (a) amino acids 29to 45 of SEQ ID NO: 194; and (b) amino acids 336 to 347 of SEQ ID No:194.
 4. The isolated antibody or antigen-binding fragment thereof ofclaim 2, wherein the antibody or antigen-binding fragment thereofinteracts with an amino acid sequence selected from the group consistingof (a) amino acids 29 to 45 of SEQ ID NO: 194; and (b) amino acids 331to 347 of SEQ ID No: 194, in the presence of ANP.
 5. The isolatedantibody or antigen-binding fragment thereof of claim 2, wherein theantibody or antigen-binding fragment thereof interacts with an aminoacid sequence selected from the group consisting of (a) amino acids 29to 45 of SEQ ID NO: 194; (b) amino acids 70 to 81 of SEQ ID NO: 194; and(c) amino acids 331 to 335 of SEQ ID No: 194, in the presence of ANP. 6.The antibody or antigen-binding fragment thereof of claim 1, wherein theantibody is a fully human monoclonal antibody.
 7. The antibody orantigen-binding fragment thereof of claim 1, wherein the antibody hasone or more properties selected from the group consisting of: (a) is afully human monoclonal antibody; (b) binds to monomeric human NPR1 inthe absence of ANP and/or brain natriuretic peptide (BNP) at 25° C. andat 37° C. with a dissociation constant (K_(D)) of less than 690 nM, asmeasured in a surface plasmon resonance assay; (c) binds to dimerichuman NPR1 in the absence of ANP or BNP at 25° C. and at 37° C. with aK_(D) of less than 42 nM, as measured in a surface plasmon resonanceassay; (d) binds to human NPR1 complexed to ANP at 25° C. and 37° C.with a K_(D) of less than 80 nM, as measured in a surface plasmonresonance assay; (e) binds to human NPR1 complexed to BNP at 25° C. and37° C. with a K_(D) of less than 20 nM, as measured in a surface plasmonresonance assay; (f) binds to monomeric monkey NPR1 in the absence ofANP and/or BNP at 25° C. and 37° C. with a K_(D) of less than 365 nM, asmeasured in a surface plasmon resonance assay; (g) binds to dimericmonkey NPR1 in the absence of ANP or BNP at 25° C. and at 37° C. with aK_(D) of less than 30 nM, as measured in a surface plasmon resonanceassay; (h) binds to monkey NPR1 complexed to ANP at 25° C. and 37° C.with a K_(D) of less than 10 nM, as measured in a surface plasmonresonance assay; (i) binds to monkey NPR1 complexed to BNP at 25° C. and37° C. with a K_(D) of less than 10 nM, as measured in a surface plasmonresonance assay; (j) does not bind to mouse NPR1; (k) binds to cellsexpressing human NPR1 (without ANP) or NPR1-complexed to ANP with a EC₅₀less than 5 nM; (l) activates NPR1 with a EC₅₀ of less than 385 nM, asmeasured in a calcium flux cell-based bioassay; (m) reduces the systemicblood pressure when administered to normotensive and hypertensive mice,wherein the reduction in systemic and mean arterial blood pressureslasts for up to 28 days upon administration of a single dose; and (n)improves glucose tolerance when administered to diet-induced obese mice.8. The antibody or antigen-binding fragment thereof of claim 1, whereinthe antibody or antigen-binding fragment thereof comprises three heavychain complementarity determining regions (CDRs) (HCDR1, HCDR2 andHCDR3) contained within a heavy chain variable region (HCVR); and threelight chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chainvariable region (LCVR), wherein the HCVR comprises: (i) an amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 18, 34,50, 66, 82, 98, 114, 130, 146, 162, and 178; (ii) an amino acid sequencehaving at least 90% identity to the amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130,146, 162, and 178; (iii) an amino acid sequence having at least 95%identity to the amino acid sequence selected from the group consistingof SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178;or (iv) an amino acid sequence selected from the group consisting of SEQID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178, saidamino acid sequence having no more than 12 amino acid substitutions, andthe LCVR comprises: (a) an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154,170, and 186; (b) an amino acid sequence having at least 90% identity tothe amino acid sequence selected from the group consisting of SEQ IDNOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and 186; (c) anamino acid sequence having at least 95% identity to the amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 74, 90, 106, 122, 138, 154, 170, and 186; or (d) an amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 74, 90, 106, 122, 138, 154, 170, and 186, said amino acid sequencehaving no more than 10 amino acid substitutions.
 9. The antibody orantigen-binding fragment thereof of claim 8 comprising a HCVR having anamino acid sequence selected from the group consisting of SEQ ID NOs: 2,18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and
 178. 10. The antibody orantigen-binding fragment thereof of claim 8 comprising a LCVR having anamino acid sequence selected from the group consisting of SEQ ID NOs:10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and
 186. 11. Theantibody or antigen-binding fragment thereof of claim 8 comprising threeCDRs contained within a HCVR selected from the group consisting of SEQID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178; andthree CDRs contained within a LCVR selected from the group consisting ofSEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and 186.12. The antibody or antigen-binding fragment of claim 11 comprising aHCVR/LCVR amino acid sequence pair selected from the group consisting ofSEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154, 162/170, and 178/186.
 13. The antibody orantigen-binding fragment thereof of claim 8 comprising: (a) a HCDR1domain having an amino acid sequence selected from the group consistingof SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, and 180;(b) a HCDR2 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150,166, and 182; (c) a HCDR3 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104,120, 136, 152, 168, and 184; (d) a LCDR1 domain having an amino acidsequence selected from the group consisting of SEQ ID NOs: 12, 28, 44,60, 76, 92, 108, 124, 140, 156, 172, and 188; (e) a LCDR2 domain havingan amino acid sequence selected from the group consisting of SEQ ID NOs:14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, and 190; and (f) aLCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160,176, and
 192. 14. The antibody or antigen-binding fragment thereof ofclaim 13 comprising CDRs selected from the group consisting of: (a) SEQID NOs: 4, 6, 8, 12, 14, and 16; and (b) SEQ ID NOs: 68, 70, 72, 76, 78,and
 80. 15. The antibody or antigen-binding fragment thereof of claim 14comprising a HCVR/LCVR amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 2/10, and 66/74.
 16. An antibody orantigen-binding fragment thereof that competes for binding tonatriuretic peptide receptor 1 (NPR1) protein with an antibody orantigen-binding fragment thereof of claim
 15. 17. An antibody orantigen-binding fragment thereof that binds to the same epitope as anantibody or antigen-binding fragment thereof of claim
 15. 18. Apharmaceutical composition comprising an isolated antibody orantigen-binding fragment thereof that binds to natriuretic peptidereceptor 1 (NPR1) protein according to claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 19. An isolated polynucleotide moleculecomprising a polynucleotide sequence that encodes a HCVR and/or LCVR ofan antibody as set forth in claim
 8. 20. A vector comprising thepolynucleotide sequence of claim
 19. 21. A host cell expressing thevector of claim
 20. 22. A method of producing an anti-NPR1 antibody orantigen-binding fragment thereof, comprising growing the host cell ofclaim 21 under conditions permitting production of the antibody orfragment, and recovering the antibody or fragment so produced.
 23. Themethod of claim 22, further comprising formulating the antibody orantigen-binding fragment thereof as a pharmaceutical compositioncomprising an acceptable carrier.
 24. A method of treating, preventingor ameliorating at least one symptom or indication of a NPR1-associateddisease or disorder, the method comprising administering apharmaceutical composition comprising a therapeutically effective amountof an antibody or antigen-binding fragment thereof of claim 1 to asubject in need thereof.
 25. The method of claim 24, wherein theNPR1-associated disease or disorder is selected from the groupconsisting of hypertension, heart failure, obesity, renal failure,chronic kidney disease, macular edema, glaucoma, stroke, lung disorders,pulmonary fibrosis, inflammation, asthma, skeletal growth disorders,bone fractures, diabetes, and cancer.
 26. The method of claim 24,wherein the pharmaceutical composition is administered prophylacticallyor therapeutically to the subject in need thereof.
 27. The method ofclaim 24, wherein the pharmaceutical composition is administered incombination with a second therapeutic agent.
 28. The method of claim 27,wherein the second therapeutic agent is selected from the groupconsisting of an aldosterone antagonist, an alpha-adrenergic blocker, anangiotensin converting enzyme (ACE) inhibitor, an arteriolarvasodilator, an autonomic ganglionic vasodilator, a beta-adrenergicblocker, a catecholamine-depleting sympatholytic, a central alpha-2adrenergic agonist, a calcium channel blocker, a diuretic, a renininhibitor, an anti-coagulant, an anti-platelet agent, a cholesterollowering agent, a vasodilator, digitalis, surgery, an implantabledevice, anti-tumor therapy, insulin, a GLP1 agonist, metformin,dialysis, bone marrow stimulant, hemofiltration, a lifestylemodification, and a dietary supplement.
 29. The method of claim 24,wherein the pharmaceutical composition is administered subcutaneously,intravenously, intradermally, intraperitoneally, or intramuscularly.